Methods for raising pre-adult anadromous fish

ABSTRACT

The invention relates to methods, compositions and kits for improving the raising of pre-adult anadromous fish, or preparing pre-adult anadromous fish for transfer to seawater. The methods involve adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to the freshwater in an amount sufficient to increase expression and/or sensitivity of at least one PVCR; and adding feed for fish consumption to the freshwater, wherein the feed comprises an amount of NaCl sufficient to contribute to a significantly increased level of the PVCR modulator in serum of the pre-adult anadromous fish.

RELATED APPLICATION(S)

[0001] This application is a divisional of U.S. application Ser. No.09/687,477, filed Oct. 12, 2000. The entire teachings of theaforementioned application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] In nature, many anadromous fish live most of their adulthood inseawater, but swim upstream to freshwater for the purpose of breeding.As a result, anadromous fish hatch from their eggs and are born infreshwater. As these fish grow, they swim downstream and gradually adaptto the seawater.

[0003] Fish hatcheries have experienced difficulty in raising thesetypes of fish because the window of time in which the pre-adult fishadapts to seawater (e.g., undergoes smoltification) is short-lived, andcan be difficult to pinpoint. As a result, these hatcheries experiencesignificant morbidity and mortality when transferring anadromous fishfrom freshwater to seawater. Additionally, many of the fish that dosurvive the transfer from freshwater to seawater are stressed, andconsequently, experience decreased feeding, and increased susceptibilityto disease. Therefore, these anadromous fish often do not grow wellafter they are transferred to seawater.

[0004] The aquaculture industry loses millions of dollars each year dueto problems it encounters in transferring pre-adult anadromous fish fromfreshwater to seawater. Hence, a need exists to improve methods involvedin transferring pre-adult anadromous fish to seawater. A further needexists to increase survival and growth, and reduce stress, of pre-adultanadromous fish that have been transferred to seawater.

SUMMARY OF THE INVENTION

[0005] The present invention relates to methods for improving theraising of pre-adult anadromous fish or preparing these fish fortransfer to seawater by increasing expression of a receptor, referred toas the Polyvalent Cation Sensing Receptor (PVCR). The expression and/orsensitivity of the PVCR is increased by subjecting the pre-adultanadromous fish to at least one modulator of the PVCR. The pre-adultanadromous fish are subjected to the modulator when it is added to theirfreshwater environment, and optionally, to the feed. The inventionencompasses adding at least one PVCR modulator to the freshwater, andadding feed for fish consumption to the freshwater. The feed containssodium chloride (NaCl) and, optionally, at least one PVCR modulator inan amount to contribute to a significantly increased level of the PVCRmodulator in the serum of the pre-adult anadromous fish. Increasedexpression and/or sensitivity of the PVCR is maintained until the fishare ready to be transferred to seawater. The pre-adult anadromous fishcan be maintained in the freshwater having at least one PVCR agonistuntil they are ready to be transferred to seawater.

[0006] In one embodiment of the invention, pre-adult anadromous fish(e.g., salmon, trout and arctic char) are prepared for transfer fromfreshwater to seawater by adding PVCR agonists, such as calcium andmagnesium to the freshwater, and adding feed for fish consumption havingbetween about 1% and about 10% NaCl by weight (e.g. between about 10,000mg/kg and 100,000 mg/kg) to the freshwater. The amount of calcium addedto the freshwater is an amount sufficient to bring the concentration upto between about 2.0 mM and about 10.0 mM, and the amount of magnesiumadded is an amount sufficient to bring the concentration up to betweenabout 0.5 mM and about 10.0 mM. The feed can optionally include a PVCRagonist, such as an amino acid. A particular amino acid that can beadded is tryptophan in an amount between about 1 gm/kg and about 10gm/kg. The present invention also includes, optionally, exposing thepre-adult anadromous fish to a photoperiod. Preferably, the photoperiodis continuous (e.g., for a continuous period of between about 12 hoursand about 24 hours in a 24 hour period).

[0007] Additional embodiments of the invention include methods ofincreasing or improving food consumption before and/or after seawatertransfer, increasing growth, increasing survival and/or reducingmortality, reducing osmotic damage, transferring parr (e.g., betweenabout 10 and about 60 grams) to seawater, and transferring a pre-adultanadromous fish to seawater having a temperature of about 14° C. toabout 19° C. These methods are performed by adding at least one PVCRmodulator to the freshwater, subjecting or exposing the pre-adultanadromous fish to at least one PVCR modulator, or introducing thepre-adult anadromous fish to freshwater having at least one PVCRmodulator, in an amount sufficient to increase expression and/orsensitivity of the PVCR. The methods also involve adding feed havingbetween about 1% and about 10% NaCl by weight to the freshwater andtransferring the pre-adult anadromous fish to seawater.

[0008] In other embodiments, the invention encompasses detection assaysor methods of determining whether a pre-adult anadromous fish, that aresubjected to at least one PVCR modulator and are fed with feed havingbetween about 1% and about 10% NaCl by weight, are ready for transfer toseawater, by assessing the amount of PVCR expression in the pre-adultanadromous fish. An increased level of expression and/or sensitivity, ascompared to a control (e.g., PVCR expression from a fish not subjectedto a PVCR modulator), indicates that the pre-adult anadromous fish areready for transfer to seawater. In a preferred embodiment, the assayincludes contacting an anti-PVCR antibody with a sample (e.g., gill,skin, intestine, urinary bladder, kidney, brain or muscle) underconditions sufficient for the formation of a complex between theantibody and the PVCR; and detecting the formation of the complex. Inanother embodiment the assay relates to hybridizing a nucleic acidsequence having a detectable label to the nucleic acid sequence of thePVCR of a sample taken from the pre-adult anadromous fish and detectingthe hybridization.

[0009] In yet another embodiment, the present invention relates tovarious compositions and mixtures. In particular, the invention pertainsto an aquatic food composition having a concentration of NaCl betweenabout 10,000 mg/kg and 100,000 mg/kg (e.g., about 12,000 mg/kg). Theaquatic food composition can optionally include a PVCR modulator (e.g.tryptophan in an amount between 1 gm/kg and 10 gm/kg).

[0010] The invention also embodies an aquatic mixture for providing anenvironment to improve the raising of pre-adult anadromous fish. Themixture includes at least one PVCR modulator. An example of such amixture is a calcium source, that when added to freshwater, provides aconcentration of between about 2.0 mM and about 10.0 mM; and a magnesiumsource, that when added to freshwater, provides a concentration ofbetween about 0.5 mM and 10.0 mM.

[0011] In yet another embodiment, the present invention relates to kits.In particular, the invention embodies kits for improving the raising ofpre-adult anadromous fish, that includes a PVCR modulator for additionto the freshwater and an aquatic food composition, as described herein.In another embodiment, the invention includes kits for determiningwhether a pre-adult anadromous fish are ready for transfer to seawater,after being subjected to at least one PVCR modulator and feed havingbetween about 1% and about 10% NaCl by weight. The kit includes eitheran anti-PVCR antibody, and a solid support; or a nucleic acid sequencehaving a detectable label that can hybridize to nucleic acid of anaquatic PVCR.

[0012] Surprisingly, it has been discovered that increased expressionand/or altering the sensitivity of the PVCR allows these pre-adultanadromous fish to better adapt to seawater. Until the discovery of thepresent invention, the aquaculture industry was unable to transfer thepre-adult anadromous fish to seawater without subjecting the fish tostress, death and/or disease. Unlike this practice, carrying out thesteps of the invention increases the expression and/or alters thesensitivity of the PVCR and allows for transfer of the pre-adultanadromous fish to seawater with minimal or no stress, death and/ordisease, and unexpectedly provides several benefits, such as increasedgrowth and the ability to transfer these fish to water having highertemperatures, as further described herein. The present invention resultsin one or more of the following advantages in transferring pre-adultanadromous fish to seawater: a reduction in mortality; improvement infeeding; an increase in growth; a decrease in the amount of diseasedfish; and/or a reduction in osmotic shock. The present invention alsoallows for earlier harvesting of the fish with increased flexibility inproducing fish year round. Additionally, the methods of the presentinvention can result in significant cost savings for fish hatcheries.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a diagram illustrating the partial nucleotide (SEQ IDNO:1) and amino acid (SEQ ID NO:2) sequences of the polyvalentcation-sensing receptor (PVCR) of Atlantic salmon (Salmo salar).

[0014]FIG. 2 is a diagram illustrating the partial nucleotide (SEQ IDNO:3) and amino acid (SEQ ID NO:4) sequences of the PVCR of arctic char(Salvelinus alpinus).

[0015]FIG. 3 is a diagram illustrating the partial nucleotide (SEQ IDNO:5) and amino acid (SEQ ID NO:6) sequences of the PVCR of rainbowtrout (Onchorhynchus mykiss).

[0016] FIGS. 4A-B are diagrams illustrating the alignment of the aminoacids sequences for shark kidney cation receptor (“SKCaR”) (SEQ ID NO:18), salmon (SEQ ID NO:2), arctic char (SEQ ID NO:4) and rainbow trout(SEQ ID NO:6).

[0017]FIG. 5 is a schematic diagram illustrating industry practice forsalmon aquaculture production, prior to the discovery of the presentinvention. The diagram depicts key steps in salmon production for S0 (75gram) and S1 (100 gram) smolts. The wavy symbol indicates freshwaterwhile the bubbles indicate seawater.

[0018]FIG. 6 is a graphical representation illustrating length (cm) andweight (gm) of APS Process I Smolts 50 days after ocean netpenplacement. APS Process I smolts had an average weight of 76.6 gram whenplaced seawater and were sampled after 50 days. APS Process I is definedis Example 2.

[0019]FIG. 7 is a graphical representation illustrating length (cm) andweight (gm) of representative APS Process I smolts prior to transfer toseawater.

[0020]FIG. 8 is a graphical representation illustrating length (cm) andweight (gm) of APS Process I smolts before transfer, and mortalities.

[0021]FIG. 9 is a three dimensional graph illustrating the survival over5 days of Arctic Char in seawater after being maintained in freshwater,APS Process I for 14 days, and APS Process I for 30 days.

[0022]FIG. 10 is a graphical representation illustrating the length (cm)and weight (gm) of St. John/St. John APS Process II smolts prior toseawater transfer. APS Process II is defined in Example 2.

[0023]FIGS. 11A and 11B are graphical representations illustratingweight (gm) and length (cm) of APS Process II smolt survivors andmortalities 5 days after transfer to seawater tanks, and 96 hours aftertransfer to ocean netpens.

[0024]FIG. 12 is a graphical representation showing weight (gm) andlength (cm) of APS Process II smolt mortalities after 5 days aftertransfer to ocean netpens.

[0025] FIGS. 13A-G are photographs of immunocytochemistry of epitheliaof the proximal intestine of Atlantic Salmon illustrating PVCRlocalization and expression.

[0026]FIG. 14 is a photograph of a Western Blot of intestinal tissuefrom salmon maintained subjected to APS Process I for immune (lanemarked CaR, e.g., a PVCR) and preimmune (land marked preimmune)illustrating PVCR expression.

[0027]FIG. 15 is a photograph of a Western Blot of intestinal tissuefrom trout fingerlings for immune (lane marked CaR, e.g., a PVCR) andpreimmune.(lane marked preimmune) illustrating PVCR expression.

[0028] FIGS. 16A-H are photographs of immunocytochemistry of epitheliaof proximal intestine of rainbow trout using anti-PVCR antiserumillustrating PVCR localization and expression.

[0029]FIG. 17 is a photograph of a Western Blot comparing levels of PVCRof fish in freshwater, water having calcium and magnesium, and seawater,illustrating PVCR expression.

[0030] FIGS. 18A-C are photographs of immunolocalization of the PVCR inthe epidermis of salmon illustrating PVCR localization and expression.

[0031]FIG. 19 is a schematic drawing illustrating adaptive changes offish in seawater and in freshwater.

[0032]FIG. 20 is a graphical representation of serum calciumconcentrations (mM) over time in rainbow trout subjected to transfer toeither seawater or water mixture of the present invention. All datapoints represent a least 5 independent determinations mean±standarddeviation from a single representative experiment.

[0033]FIG. 21 is a graphical representation showing increases in serumcalcium concentrations (mM) over time induced by feeding troutmaintained in a water mixture (3 mM calcium, 1 mM magnesium) and astandard freshwater pelleted diet containing additional 1% sodiumchloride (w/w).

[0034]FIGS. 22A and 22B are graphical representations of alterations inserum calcium (FIG. 22A) and sodium (FIG. 22B) after seawater transferof S1 Altantic salmon smolts.

[0035]FIGS. 23A and B are graphical representations of serum calcium,magnesium and sodium levels (mM) over time from Atlantic Salmon S1 APSProcess I treated fish. Each value displays the mean+/−S.D. of a minimumof 10 independent determination from this single representativeexperiment.

[0036]FIG. 24 is a graphical representation illustrating the weight (gm)and length (cm) of representative APS Process II smolts prior totransfer to seawater. This representative sample (n=100) of APS ProcessII smolts possess a wide range of body weights (3.95-23 gram) with anaverage body weight of 11.5 gm. Note that all mortalities (n=10)occurred only in the smaller fish in the transfer group.

[0037]FIG. 25 is a graphical representation illustrating thequantitation of serum concentrations (mM) of calcium, magnesium andsodium in preadult Atlantic salmon subjected to APS Process II aftertheir transfer to seawater. All values shown are the mean+S.D. of aminimum of 10 independent samples from a single representativeexperiment.

[0038] FIGS. 26A-C are an alignment illustrating nucleic acid sequencesfor the PVCR of Atlantic Salmon (SEQ ID NO.: 1), Char (SEQ ID NO.: 3),Chum Salmon (SEQ ID NO.:7), Coho Salmon (SEQ ID NO.:9), King Salmon (SEQID NO.:11), Pink Salmon (SEQ ID NO.:13), Sockeye Salmon (SEQ ID NO.:15)and Trout (SEQ ID NO.: 5).

[0039] FIGS. 27A-B are an alignment illustrating the open reading frameof the polypeptide sequences for the PVCR of Atlantic Salmon (SEQ IDNO.: 2), Char (SEQ ID NO.: 4), Chum Salmon (SEQ ID NO.: 8), Coho Salmon(SEQ ID NO.: 10), King Salmon (SEQ ID NO.: 12), Pink Salmon (SEQ ID NO.:14), Sockeye Salmon (SEQ ID NO.: 16) and Trout (SEQ ID NO.: 6).

[0040] FIGS. 28A-B are a diagram illustrating the nucleic acid sequenceof SKCaR (SEQ ID NO.: 17).

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to methods for improving theraising of pre-adult anadromous fish and/or methods for preparingpre-adult anadromous fish for transfer from freshwater to seawater. Themethods involve increasing expression and/or altering the sensitivity ofa Polyvalent Cation Sensing Receptor (PVCR) (e.g., at least one PVCR).The invention relates to increasing expression of the PVCR that affectsthe fish's ability to adapt to seawater (e.g., to smolt), to undergosmoltification, to survive, to increase growth, to increase foodconsumption, and/or to be less susceptible to disease.

[0042] In particular, the methods of the present invention includeadding at least one PVCR modulator to the freshwater, and adding aspecially made or modified feed to the freshwater for consumption by thefish. The feed contains a sufficient amount of sodium chloride (NaCl)(e.g., between about 1% and about 10% by weight, or about 10,000 mg/kgto about 100,000 mg/kg) to significantly increase levels of the PVCRmodulator in the serum. This amount of NaCl in the feed causes orinduces the pre-adult anadromous fish to drink more freshwater. Sincethe freshwater contains a PVCR modulator and the fish ingest increasedamounts of it, the serum level of the PVCR modulator significantlyincreases in the fish, and causes increased PVCR expression and/oraltered PVCR sensitivity. This process allows the pre-adult anadromousfish to be “pre-conditioned” and better adapt to seawater.

[0043] The methods of the present invention pertain to adaptingpre-adult anadromous fish to seawater. Anadromous fish are fish thatswim from seawater to freshwater to breed. Anadromous fish include, forexample, salmon (e.g, Atlantic Salmon (Salmo salar), Coho Salmon(Oncorhynchus kisutch), Chinook Salmon (Oncorhynchus tshawytscha), ChumSalmon (Oncorhynchus keta), Pink Salmon (Oncorhynchus gorbuscha)), char(e.g., Arctic Char (Salveninus alpinus)) and trout (e.g., Rainbow Trout(Oncorhynchus mykiss)). Anadromous fish also include fish that areunable to swim to seawater (e.g., landlocked), but have thephysiological mechanisms to adapt to seawater. The term “pre-adultanadromous fish,” as used herein, refers to anadromous fish that havenot yet adapted to seawater. These fish are generally juvenile fish.Pre-adult anadromous fish include, but are not limited to fish that arefingerlings, parr or smolts. As used herein, a “smolt” is a fishundergoing physiological changes that allows the fish to adapt toseawater, or survive upon subsequent transfer to seawater. The term,“smolt,” also refers to a fish that is not at the precise developmentalstage to survive uninjured upon transfer to seawater, but rather is oneof a population of fish wherein, based on a statistical sampling andevaluation, the population of fish is determined to be at aphysiological stage ready for transfer to seawater.

[0044] The present invention includes methods for preparing pre-adultanadromous fish undergoing the process of smoltification for transfer toseawater. Smoltification is the stage at which a fish undergoes theacclimation or adaptation from freshwater to seawater. Smoltificationalso refers to a process occurring in pre-adult anadromous fish that isphysiological pre-adaption to seawater while still in freshwater. Thesmolification process varies from species to species. Different speciesof anadromous fish can undergo smoltification at different sizes,weights, and times in the life of the fish. The present inventioninduces the vast majority or all of the pre-adult anadromous fish toundergo this process and be prepared for transfer to seawater.

[0045] The pre-adult anadromous fish are maintained in freshwater priorto adding the PVCR modulator. The term, “freshwater,” means water thatcomes from, for example, a stream, river, ponds, public water supply, orfrom other non-marine sources having, for example, the following ioniccomposition: less than about 2 mM of magnesium, calcium and NaCl. Theterm “freshwater” also refers to freshwater to which at least one PVCRmodulator has been added, as described herein.

[0046] The PVCR modulator is added to the freshwater in sufficientamounts to increase expression or alter the sensitivity of the PVCR. APVCR has been isolated from various tissue of several types ofanadromous fish using molecular biology techniques, as described inExample 9. DNA was isolated from muscle samples from various species ofanadromous fish including Atlantic Salmon, Char, Chum Salmon, CohoSalmon, King or Chinook Salmon, Pink Salmon, Sockeye Salmon and Trout.The DNA was amplified using polymerase Chain Reaction (PCR) methodology.The amplified DNA was purified and subcloned into vectors, and theirsequences were determined, as described in Example 9.

[0047] The PVCR, which is located in various tissues (e.g., gill, skin,intestine, kidney, urinary bladder, brain or muscle) of the pre-adultanadromous fish, senses alterations in PVCR modulators including variousions (e.g., divalent cations), for example, in the surrounding water, intheir serum or in the luminal contents of tubules inside the body, suchas kidney, urinary bladder, or intestine. Its ability to sense thesemodulators increases expression of the PVCR, thereby allowing the fishto better adapt to seawater. Increased expression of the PVCR can occur,for example, in one or all tissues.

[0048] A “PVCR modulator” is defined herein to mean a compound whichincreases expression of the PVCR, or increases the sensitivity orresponsiveness of the PVCR. Such compounds include, but are not limitedto, PVCR agonists (e.g., inorganic polycations, organic polycations andamino acids), Type II calcimimetics, and compounds that indirectly alterPVCR expression (e.g., 1,25 dihydroxyvitamin D in concentrations ofabout 3,000-10,000 International Units/kg feed), cytokines such asInterleukin Beta, and Macrophage Chemotatic Peptide-1 (MCP-1)). Examplesof Type II calcimimetics, which increase expression and/or sensitivityof the PVCR, are, for example, NPS-R-467 and NPS-R-568 from NPSPharmaceutical Inc., (Salt Lake, Utah, U.S. Pat. Nos. 5,962,314;5,763,569; 5,858,684; 5,981,599; 6,001,884) which can be administered inconcentrations of between about 0.1 μM and about 100 μM feed or water.See Nemeth, E. F. et al., PNAS 95: 4040-4045 (1998). Examples ofinorganic polycations are divalent cations including calcium at aconcentration between about 2.0 and about 10.0 mM and magnesium at aconcentration between about 0.5 and about 10.0 mM; and trivalent cationsincluding, but not limited to, gadolinium (Gd3+) at a concentrationbetween about 1 and about 500 μM. Organic polycations include, but notlimited to, aminoglycosides such as neomycin or gentamicin inconcentrations of between about 1 and about 8 gm/kg feed as well asorganic polycations including polyamines (e.g., polyarginine,polylysine, polyhistidine, polyornithine, spermine, cadaverine,putricine, copolymers of poly arginine/histidine, poly lysine/argininein concentrations of between about 10 μM and 10 mM feed). See Brown, E.M. et al., Endocrinology 128: 3047-3054 (1991); Quinn, S. J. et al., Am.J. Physiol. 273: C1315-1323 (1997). Additionally, PVCR agonists includeamino acids such as L-Tryptophan L-Tyrosine, L-Phenylalanine, L-Alanine,L-Serine, L-Arginine, L-Histidine, L-Leucine, L-Isoleucine, andL-Cystine at concentrations of between about 1 and about 10 gm/kg feed.See Conigrave, A. D., et al., PNAS 97: 4814-4819 (2000). The molarconcentrations refer to free or ionized concentrations of the PVCRmodulator in the freshwater, and does not include amounts of bound PVCRmodulator (e.g., PVCR modulator bound to negatively charged particlesincluding glass, proteins, or plastic surfaces). Any combination ofthese modulators can be added to the water or to the feed (in additionto the NaCl, as described herein), so long as the combination increasesexpression and/or sensitivity of the PVCR.

[0049] The PVCR modulator can be administered to the fish in a number ofways. The invention encompasses administration of the PVCR in any waythat is sufficient to increase the expression and/or alter thesensitivity of the PVCR. In one embodiment, the PVCR modulator is simplyadded to the freshwater in various concentrations, as described herein.A freshwater environment having at least one PVCR modulator is referredto herein as a “PVCR modulator environment.” PVCR modulators that areadded to the water increase expression and/or alter the sensitivity ofthe PVCR on the skin and gills of the fish, and can be ingested by thefish, in particular, when fish are fed feed having between about 1% andabout 10% NaCl (e.g., in concentrations between about 1 and about 10gm/100 gm feed). In addition to adding NaCl to the feed, the PVCRmodulator can also be added to the feed. Amounts and types of PVCRmodulators added to the feed are also described herein. Otherembodiments include subjecting the fish to the PVCR modulator by“dipping” the fish in the modulator, e.g., organic polycations. Theorganic polycations can be formulated in such a way as to allow thepolycations to adhere to the skin and gills of the fish, in sufficientamounts to increase expression of the PVCR.

[0050] In one preferred embodiment, the present invention is practicedby adding a combination of two PVCR agonists to the freshwater. Inparticular, calcium and magnesium are added to the freshwater to bringthe concentrations of each to between about 2.0 mM and about 10.0 mM ofcalcium, and between about 0.5 mM and about 10.0 mM of magnesium. Inaddition to adding calcium and magnesium to the water, these ranges ofion concentrations can be achieved by providing a brackish water (e.g.,diluted seawater) environment for the fish.

[0051] Calcium and magnesium can come from a variety of sources, thatwhen added to the water, the calcium and/or magnesium levels increaseexpression of the PVCR, and/or are within the stated ranges. Sources ofcalcium and magnesium can be a mixture of a variety of compounds, oreach can come from a substantially uniform or pure compound. Sources ofcalcium include, for example, Ca(CO₃)₂, CaCl₂, CaSO₄, and Ca(OH)₂ andsources of magnesium include, for example, MgCl₂, MgSO₄, MgBr₂, andMgCO₃.

[0052] In one embodiment, the invention includes intermittent (e.g.,interrupted) as well as continuous (e.g., non-interrupted) exposure tofreshwater having at least one PVCR modulator, while on the NaCl diet.Intermittent exposure to the PVCR can occur so long as the PVCRexpression and/or altered sensitivity remains increased. Continuousmaintenance in or exposure to freshwater having at least one PVCRmodulator is shown in Examples 2 and 7.

[0053] The process of the present invention pre-conditions the fish andprepares them for transfer. The pre-adult anadromous fish are maintainedin a freshwater environment having a PVCR modulator long enough toincrease the expression and/or alter sensitivity of the PVCR. The lengthof time depends on the physiological and physical maturity of the fish.Some fish will more readily adapt to the environment, and increase theirexpression and/or alter the sensitivity of their PVCR, while others willneed more time to do so. Factors that can influence the length of timenecessary to increase the expression and/or alter sensitivity of thePVCR include, but are not limited to, size of the fish, level of PVCRexpression or sensitivity, if any, prior to addition of the PVCRmodulator to the freshwater, the fish's ability to excrete the PVCRmodulator and ions, the fish's surface to volume ratio, etc. Therefore,the length of time the fish is maintained can range from about 7 days toseveral months (e.g., 7, 14, 21, 30, 45, 90 and 120 days). The fish canalso be maintained indefinitely so long as the fish are maintained infreshwater having the PVCR modulator and being fed a NaCl diet. Forexample, salmon, trout or char weighing less than 10 gms can bemaintained in freshwater having a PVCR modulator, and fed a NaCl dietfor at least about 180 days, prior to transfer to seawater.

[0054] The invention further includes adding feed to the freshwater. Thefrequency and amounts of feed that fish are fed, are taught in the art.Generally, the fish are fed 1-3 times a day, totaling about 0.25-5.0%body weight/day. The feed has enough NaCl to contribute to a significantincreased level of the PVCR modulator in the serum of the pre-adultanadromous fish. More specifically, NaCl has at least two effects. Thefirst occurs when sufficient amounts of NaCl is present in the feed. Thepresence of NaCl in the feed causes the pre-adult anadromous fish todrink more water from the surrounding environment. Second, NaCl is adirect negative PVCR modulator, and works to decreases PVCR sensitivity.Despite NaCl's effect in decreasing sensitivity, it surprisinglyincreases PVCR expression when fish are fed a NaCl diet and thesurrounding freshwater environment has at least one PVCR modulator itin. The increase in the ingestion of freshwater having PVCR modulatorscauses an overall increase of the serum levels of PVCR modulators.

[0055] The present invention also relates to an aquatic foodcomposition. The feed contains between about 1%-10% of NaCl by weight,or between about 10,000 mg of NaCl/kg of feed and about 100,000 mg ofNaCl/kg of feed (e.g., 12,000 mg/kg). The feed is referred to herein asa “NaCl diet.” The NaCl can be combined with other sodium salts toconfer the desired effect of increasing PVCR expression, altering PVCRsensitivity and/or inducing the fish to drink more. Hence, as usedherein, the term NaCl, includes a substantially pure compound, andmixtures of NaCl with other sources of sodium. The feed can furtherinclude a PVCR modulator, and in particular a PVCR agonist such as anamino acid. In one embodiment, the feed has between about 1% and about10% NaCl by weight and an amino acid such as tryptophan in an amountbetween about 1 and about 10 gm/kg.

[0056] The feed can be made in a number of ways, so long as the properconcentration of NaCl is present. The feed can be made, for example, byreformulating the feed, or by allowing the feed to absorb a solutionhaving the NaCl and optionally, adding a PVCR modulator. A top dressingcan be added for palatability. Example 8 describes in detail one way tomake the feed.

[0057] Another embodiment of the present invention includes feedingpre-adult anadromous fish feed having between 1% and 10% NaCl by weightwhen the fish are maintained in a freshwater environment having betweenabout 2.0 and about 10.0 mM of calcium, and between about 0.5 mM andabout 10.0 mM of magnesium. When this embodiment of the presentinvention is carried out, the levels of calcium, magnesium and/or sodiumin the serum of the pre-adult anadromous fish increases, as compared toidentically paired fish maintained in freshwater, between about 1% and60%, between about 1% and 40%, and between about 1% and 15%,respectively.

[0058] In another embodiment, the fish, while in the freshwater havingthe PVCR modulator, are also exposed to a photoperiod. A photoperiodrefers to exposing the fish to light (e.g., sunlight, incandescent lightor fluorescent light). Preferably, the photoperiod is substantiallycontinuous, or occurs long enough to increase growth and/or inducesmotification. The photoperiod can occur for at least about 12 hourswithin a 24 hour interval, or for longer periods such as about 14, 16,18, 20, 22 or preferably, about 24 hours.

[0059] After being exposed to the steps of the present invention, thepre-adult anadromous fish are transferred to seawater. The term,“seawater,” means water that comes from the sea, or water which has beenformulated to simulate the chemical and mineral composition of waterfrom the sea. The major elemental composition of the prepared seawaterpreferably falls substantially within the range of the major elementalcomposition of the natural seawater (e.g., having the following ioniccomposition: greater than 30 mM of magnesium, greater than about 6 mM ofcalcium, and greater than about 300 mM NaCl). Methods of preparingartificial seawater are known in the art and are described in, forinstance, U.S. Pat. No. 5,351,651.

[0060] When performing the methods of the present invention on pre-adultanadromous fish, the fish exhibit significant increased growth and foodconsumption, as compared to pre-adult anadromous fish that are notsubjected to the present invention. Upon transfer to seawater, fish thatare not subjected to the steps of the present invention generallyexperience osmotic stress, reduced or no food consumption, and evendeath. Osmotic stress results from differences in the osmotic pressurebetween the surrounding environment and body compartments of the fish.This disturbs the homeostatic equilibrium of the fish and results indecreased growth, reproductive failure and reduced resistance todisease. The fish that have been preconditioned by the steps of thepresent invention do not experience a significant amount of osmoticstress, and begin feeding on or soon after transfer to seawater. As aresult, the fish also grow earlier. In particular, pre-adult anadromousfish that ingested a feed having between about 1% and about 10% NaCl,and between about 1 gm and about 10 gms per kg of feed of an amino acid,exhibit a substantial increase in growth. In the experiments, the fishadapted by the present invention have shown as much as about 65%increased growth during the same interval of time, as compared toidentically paired fish that did not undergo the steps of the presentinvention and were transferred to seawater. See Table 4 of Example 2.Elimination of low feeding or poorly feeding osmotically stressed fishin a group improves the overall feed conversion ratio of the entiregroup. Optimal feeding and growth after seawater transfer by all membersof the group of pre-conditioned fish will permit better feed utilizationand improve the overall yield of production when fish reach market size.

[0061] Similarly, the present invention allows for decreasing orreducing the time between generations of pre-adult anadromous fish.These fish begin breeding earlier because the present inventionincreases their growth, as described herein. Since 2-3 years arerequired to obtain sexually mature fish, attempts to engage in selectivebreeding of traits requires this 2-3 year interval before a given traitcan be selected for and the fish exhibit that trait breed. Improvementsin growth and time to reach maturity produced by the invention reducethe time interval required to reach sexual maturity in fish by as muchas about 6 months to about 12 months. Reducing the interval for breedingallows for the production of more fish, and the improved selection offish that possess traits other than those that are better able to adaptto seawater (e.g., select for fish that have improved taste, increasedfilet thickness, increased α3 omega fatty acid content, or fish that aremore readily able to increase PVCR expression).

[0062] Prior to the present invention, anadromous fish that aretransferred from freshwater to seawater are generally transferred at aparticular size, referred to as “critical size.” The critical sizevaries from species to species, but generally refers to a minimum sizeat which a fish can be transferred to seawater. The critical size forsalmon, trout and char is between about 50 and about 100 gms, betweenabout 70 and about 120 gms, and greater than 100 gms, respectively.Critical sizes for Coho, King, and Sockeye Salmon are between about 10and about 15 gms, between about 20 and 40 gms and between about 1 andabout 2 gms, respectively. Chum and Pink Salmon each have a criticalsize about less than 3 gms.

[0063] Prior to the invention, a population of pre-adult anadromous fishhaving attained a mean critical size were transferred to seawater. Someof the fish are physiologically ready for the transfer, while others arenot. This is one of the reasons for the increased mortality rate upontransfer to seawater. The methods of the present inventionphysiologically prepares all or mostly all of the fish for transfer toseawater by increasing PVCR expression and/or sensitivity, and/or byinducing smoltification. Greater than about 80% (e.g., 90%, 95%, 100%)undergo smoltification and are ready for transfer to seawater. In fact,in one experiment, when performing the steps of the present invention onAtlantic Salmon (e.g., subjecting the fish to a PVCR modulatorenvironment and a NaCl diet), close to 100% of the Atlantic Salmonunderwent smoltification. See Example 2. Hence, the methods of thepresent invention include methods of preparing pre-adult anadromous fishfor transfer to seawater, as well as inducing smotification in pre-adultanadromous fish.

[0064] Since the methods of the present invention increase theexpression and/or sensitivity of the PVCR in pre-adult anadromous fish,they survive better when transferred to seawater. The reduced osmoticstress results in reduced mortality. In one case, certain populations ofpre-adult anadromous fish that did not undergo the methods of thepresent invention exhibit a 100% mortality rate after transfer toseawater (see FIG. 9, Example 2), while other populations of pre-adultanadromous fish that did not undergo the methods of the invention havesurvival rates of only between about 40% and 70%. See Table 1, Example2. This occurs because the fish experience osmotic shock whentransferred to seawater which has a very different ionic compositionthan freshwater. However, when preconditioned by the methods of thepresent invention, the fish exhibit a survival rate that issignificantly greater than the rate for unconditioned fish (e.g.,betweenabout 80% about 100%). In fact, when performing the present invention onAtlantic Salmon, 99% of the fish survived transfer to seawater after 5days, as compared to 33% of fish that did not undergo the steps of thepresent invention in one experiment. See Table 1 of Example 2. Hence,the present invention embodies methods of reducing the mortality rateafter pre-adult anadromous fish are transferred to seawater.

[0065] Not only is the present invention useful in reducing mortalityrates after transfer to seawater, the present invention is also used toincrease survival rates in freshwater prior to transfer. Prior to thediscovery of the present invention, a “smolt window” existed in whichthe hatcheries transferred the pre-adult anadromous fish to seawater, orelse the fish will die if they continue to remain in freshwater afterthey undergo smoltification. The PVCR modulator environment and the NaCldiet of the present invention allow the fish to continue to thriveindefinitely. The fish continue to consume feed and grow. When thepresent invention was performed on Atlantic Salmon, 99% of the fishsurvived and thrived for at least 45 days in freshwater. In contrast,only 67% of the fish that did not undergo the steps of the inventionsurvived after 45 days in freshwater in one experiment. See Example 2.

[0066] The present invention also includes methods for transferring toseawater pre-adult anadromous fish having smaller weights, as comparedto the industry recognized critical size for the particular species offish. The methods of the present invention, as described herein,increase PVCR expression in fish that are smaller than those normallytransferred to seawater, or those undergoing or about to undergosmoltification. These methods include transferring a parr, the stage ofa juvenile fish prior to becoming a smolt, to seawater. Parr is a lifestage of pre-adult anadromous fish that occurs after maturation ofalevins or yolk sac fry. Parr or fingerlings display characteristic ovidstripes or parr marks along their flanks, and normally undergo growthand development in freshwater prior to smoltification. The term “parr”is a term that is known in the art. As yolk sac fry continue to feed,they grow into larger parr. Parr can possess a wide range of bodyweights depending on conditions under which they are grown. The weightsof parr vary from species to species. Body weights for parr varysignificantly with a range from about 0.5 gms to about 70 gms. Carryingout the present invention in one experiment, as described herein,results in a transfer of Atlantic Salmon parr weighing as little asbetween about 13% and about 18.5% of the critical size weight (betweenabout 70 and about 100 gms), or about 13 gms Adding a PVCR modulator tothe feed (e.g., an amino acid such as a tryptophan), in addition to theNaCl diet, allows seawater transfer of fish having particularly lowweights. See Example 2.

[0067] The present invention additionally provides methods fortransferring pre-adult anadromous fish into seawater having warmertemperatures (e.g., 14° C. and 19° C.), as compared to watertemperatures into which these fish have been transferred in the past.Since the fish experience reduced or little osmotic stress whentransferred to seawater using the methods of the present invention, thefish are able to withstand transfer into higher water temperatureswithout exhibiting an increase in mortality rates. See Example 2.

[0068] The methods of the present invention also decrease the incidenceof disease among the smolts and the growing salmon. Because smoltstreated with the methods of the present invention experience less stressupon transfer to seawater, their immune functions are stronger, and theyare less susceptible to parasitic, viral, bacterial and fungal diseases.Fish not treated with the methods described herein are more susceptibleto such diseases, and can serve as reservoirs of disease, capable ofinfecting healthy fish.

[0069] Methods Assessment of the PVCR

[0070] The present invention includes methods of detecting the level ofthe PVCR to determine whether fish are ready for transfer fromfreshwater to seawater. Methods that measure PVCR levels include severalsuitable assays. Suitable assays encompass immunological methods, suchas FACS analysis, radioimmunoassay, flow cytometry, enzyme-linkedimmunosorbent assays (ELISA) and chemiluminescence assays. Any methodknown now or developed later can be used for measuring PVCR expression.

[0071] Antibodies reactive with the PVCR or portions thereof can beused. In a preferred embodiment, the antibodies specifically bind withthe PVCR or a portion thereof. The antibodies can be polyclonal ormonoclonal, and the term antibody is intended to encompass polyclonaland monoclonal antibodies, and functional fragments thereof. The termspolyclonal and monoclonal refer to the degree of homogeneity of anantibody preparation, and are not intended to be limited to particularmethods of production.

[0072] In several of the preferred embodiments, immunological techniquesdetect PVCR levels by means of an anti-PVCR antibody (i.e., one or moreantibodies). The term “anti-PVCR” antibody includes monoclonal and/orpolyclonal antibodies, and mixtures thereof.

[0073] Anti-PVCR antibodies can be raised against appropriateimmunogens, such as isolated and/or recombinant PVCR or portion thereof(including synthetic molecules, such as synthetic peptides). In oneembodiment, antibodies are raised against an isolated and/or recombinantPVCR or portion thereof (e.g., a peptide) or against a host cell whichexpresses recombinant PVCR. In addition, cells expressing recombinantPVCR, such as transfected cells, can be used as immunogens or in ascreen for antibody which binds receptor.

[0074] Any suitable technique can prepare the immunizing antigen andproduce polyclonal or monoclonal antibodies. The art contains a varietyof these methods (see e.g., Kohler et al., Nature, 256: 495-497 (1975)and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E.and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing a suitable immortal or myeloma cell line, such asSP2/0, with antibody producing cells can produce a hybridoma. Animalsimmunized with the antigen of interest provide the antibody producingcell, preferably cells from the spleen or lymph nodes. Selective cultureconditions isolate antibody producing hybridoma cells while limitingdilution techniques produce them. Researchers can use suitable assayssuch as ELISA to select antibody producing cells with the desiredspecificity.

[0075] Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice.

[0076] According to the method, an assay can determine the level of PVCRin a biological sample. In determining the amounts of PVCR, an assayincludes combining the sample to be tested with an antibody havingspecificity for the PVCR, under conditions suitable for formation of acomplex between antibody and the PVCR, and detecting or measuring(directly or indirectly) the formation of a complex. The sample can beobtained directly or indirectly, and can be prepared by a methodsuitable for the particular sample and assay format selected.

[0077] In particular, tissue samples, e.g., gill tissue samples, can betaken from fish after they are anaesthetized with MS-222. The tissuesamples are fixed by immersion in 2% paraformaldehyde in appropriateRingers solution corresponding to the osmolality of the fish, washed inRingers, then frozen in an embedding compound, e.g., O.C.T.™ (Miles,Inc., Elkahart, Ind., USA) using methylbutane cooled with liquidnitrogen. After cutting 8-10μ tissue sections with a cryostat,individual sections are subjected to various staining protocols. Forexample, sections are: 1) blocked with goat serum or serum obtained fromthe same species of fish, 2) incubated with rabbit anti-CaR or anti-PVCRantiserum, and 3) washed and incubated with peroxidase-conjugatedaffinity-purified goat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum are then visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections are mounted, viewed and photographed by standardlight microscopy techniques. The anti-CaR antiserum used to detect fishPVCR protein is raised in rabbits using a 23-mer peptide correspondingto amino acids numbers 214-236 localized in the extracellular domain ofthe RaKCaR protein. The sequence of the 23-mer peptide is:ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO.: 19) A small peptide with thesequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 20) or ARSRNSADGRSGDDLPC(SEQ ID NO.: 21) can also be used to make antisera containing antibodiesto PVCRs. Such antibodies can be monoclonal, polyclonal or chimeric.

[0078] Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as 32P, 125I, 131I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,β-galactosidase, biotin, avidin, spin labels and the like. The detectionof antibodies in a complex can also be done immunologically with asecond antibody which is then detected (e.g., by means of a label).Conventional methods or other suitable methods can directly orindirectly label an antibody.

[0079] In performing the method, the levels of the PVCR that aredistinct from the control. Increased levels or the presence of PVCRexpression, as compared to a control, indicate that the fish or thepopulation of fish from which a statistically significant amount of fishwere tested, are ready for transfer to freshwater. A control refers to alevel of PVCR, if any, from a fish that is not subjected to the steps ofthe present invention, e.g., not subjected to freshwater having a PVCRmodulator and/or not fed a NaCl diet. For example, FIGS. 13 and 18 showthat fish not subjected to the present invention had no detectable PVCRlevel, whereas fish that were subjected to the steps of the inventionhad PVCR levels that were easily detected.

[0080] The PVCRs can also be assayed by Northern blot analysis of mRNAfrom tissue samples. Northern blot analysis from various shark tissueshas revealed that the highest degree of PVCRs expression is in gilltissue, followed by the kidney and the rectal gland. There appear to beat least three distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb.

[0081] The PVCRs can also be assayed by hybridization, e.g., byhybridizing one of the PVCR sequences provided herein (e.g., SEQ ID NO:1,3,5,7,9, 11, 13, 15) or an oligonucleotide derived from one of thesequences, to a DNA-containing tissue sample from a fish. Such ahybridization sequence can have a detectable label, e.g., radioactive,fluorescent, etc., attached, to allow to detection of hybridizationproduct. Methods for hybridization are well known, and such methods areprovided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entireteachings of which are herein incorporated by reference in theirentirety. The design of the oligonucleotide probe should preferablyfollow these parameters: (a) it should be designed to an area of thesequence which has the fewest ambiguous bases (“N's”), if any, and (b)it should be designed to have a T_(m) of approx. 80° C. (assuming 2° C.for each A or T and 4 degrees for each G or C).

[0082] Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but also onfactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (T_(m)) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the T_(m) of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

[0083] High stringency conditions can employ hybridization at either (1)1×SSC (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1×Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na₂EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated T_(m) of the hybrid, where T_(m)in ° C.=(2×the number of A and T bases)+(4×the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61(%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

[0084] Moderate stringency conditions can employ hybridization at either(1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/mldenatured calf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS,0.1-2 mg/ml denatured calf thymus DNA at 42° C., (3) 1% bovine serumalbumen (fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 MNaHPO₄=134 g Na₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2mg/ml denatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC,0.02 M Tris-HCl (pH 7.6), 1×Denhardt's solution (100×=10 g Ficoll 400,10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), waterto 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calfthymus DNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100μg/ml denatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA-DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5-10°C. below that of the calculated T_(m) of the hybrid, where T_(m) in °C.=(2×the number of A and T bases)+(4×the number of G and C bases). Forhybrids believed to be about 18 to about 49 base pairs in length, theT_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61(%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

[0085] Low stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 40° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1×Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 40° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 50° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na₂EDTA, 40 mMNaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10° C. below that of the calculated T_(m)of the hybrid, where T_(m) in ° C.=(2×the number of A and Tbases)+(4×the number of G and C bases). For hybrids believed to be about18 to about 49 base pairs in length, the T_(m) in ° C.=(81.5°C.+16.6(log₁₀M)+0.41(% G+C)−0.61(% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

[0086] Hence, the present invention includes kits for the detection ofthe PVCR or the quantification of the PVCR having either antibodiesspecific for the PVCR or a portion thereof, or a nucleic acid sequencethat can hybridize to the nucleic acid of the PVCR.

[0087] Alterations in the expression or sensitivity of PVCRs could alsobe accomplished by introduction of a suitable transgene. Suitabletransgenes would include either the PVCR gene itself or modifier genesthat would directly or indirectly influence PVCR gene expression.Methods for successful introduction, selection and expression of thetransgene in fish oocytes, embryos and adults are described in Chen, T Tet al., Transgenic Fish, Trends in Biotechnology 8:209-215 (1990).

[0088] The present invention is further and more specificallyillustrated by the following Examples, which are not intended to belimiting in any way.

EXEMPLIFICATION Example 1 Polyvalent Cation-sensing Receptors (PVCRs)Serve as Salinity Sensors in Fish.

[0089] Polyvalent cation-sensing receptors (PVCRs) serve as salinitysensors in fish. These receptors are localized to the apical membranesof various cells within the fish's body (e.g., in the gills, intestine,kidney) that are known to be responsible for osmoregulation. Afull-length cation receptor (CaR) from the dogfish shark has beenexpressed in human HEK cells. This receptor was shown to respond toalterations in ionic compositions of NaCl, Ca2+ and Mg2+ inextracellular fluid bathing the HEK cells. The ionic concentrationsresponded to encompassed the range which includes the transition fromfreshwater to seawater. Expression of PVCR mRNA is also increased infish after their transfer from freshwater to seawater, and is modulatedby PVCR agonists. Partial genomic clones of PVCRs have also beenisolated from other fish species, including winter and summer flounderand lumpfish, by using nucleic acid amplification with degenerateprimers.

[0090] This method was also used to isolate partial genomic clones ofPVCRs for Atlantic salmon (FIG. 1), arctic char (FIG. 2) and rainbowtrout (FIG. 3). The degenerate oligonucleotide primers used were 5′-TGTCKT GGA CGG AGC CCT TYG GRA TCG C-3′ (SEQ ID NO:22) AND 5′-GGC KGG RATGAA RGA KAT CCA RAC RAT GAA G-3′ (SEQ ID NO:23), where K is T or G, Y isC or T, and R is A or G. The degenerate oligos were generated bystandard methodologies (Preston, G. M., 1993, “Polymerase chain reactionwith degenerate oligonucleotide primers to clone gene family members,”in: Methods in Mol. Biol., vol. 58, ed. A. Harwood, Humana Press, pp.303-312). Genomic bands from these three species were amplified,purified by agarose gel electrophoresis, ligated into an appropriateplasmid vector (salmon and arctic char species-pT7 Blue (Novagen,Madison, Wis.; trout used pGem-T (Promega Biotech. Madison, Wis.) , andtransformed into an appropriate bacterial host strain salmon and arcticchar-pT7 vector with NovaBlue (Novagen, Madison, Wis.) and trout pGEM-Tused JM-109 E. coli cell which was then grown in liquid medium. Theplasmids and inserts were purified from the host cells, and sequenced.FIG. 4 shows the deduced amino acid sequences and alignment for thePVCRs from Atlantic salmon, arctic char and rainbow trout, relative tothe PVCR from the kidney of the dogfish shark (Squalus acanthias).

Example 2 Survival and Growth of Pre-Adult Anadromous Fish Using theMethods of the Present Invention

[0091] An important feature of current salmon farming is the placementof smolt from freshwater hatcheries to ocean netpens. Present daymethods use smolt that haave attained a critical size of approximately70-110 grams body weight. The present invention can either be utilizedboth to improve the ocean netpen transfer of standard 70-110 grams smoltas well as permit the successful ocean netpen transfer of smolt weighingonly 30 grams. For standard 70-100 gram smolt, application of theinvention eliminates the phenomenon known as “smolt window” and permitsfish to be maintained and transferred into ocean water at 15° C. orhigher. Use of the invention in 30 gram or smaller smolt permits greaterutilization of freshwater hatchery capacities followed by successfulseawater transfer to ocean netpens. In both cases, fish that undergo thesteps of the invention feed vigorously within a short interval of timeafter transfer to ocean netpens and thus exhibit rapid growth rates upontransfer to seawater.

[0092]FIG. 5 shows in schematic form the key features of currentaquaculture of Atlantic salmon in ocean temperatures present in Europeand Chile. Eggs are hatched in inland freshwater hatcheries and theresulting fry grow into fingerlings and parr. Faster growing parr areable to undergo smoltification and placement in ocean netpens as S0smolt (70 gram) during year 01. In contrast, slower growing parr aresmoltified in year 02 and placed in netpens as S1 smolt (100 gram). Inboth S0 and S1 transfers to seawater, the presence of cooler ocean andfreshwater temperatures are desired to minimize the stress of osmoticshock to newly transferred smolt. This is particularly true for S1 smoltsince freshwater hatcheries are often located at significant distancesfrom ocean netpen growout sites and their water temperatures riserapidly during early summer. Thus, the combination of rising watertemperatures and the tendency of smolt to revert or die when held forprolonged intervals in freshwater produces a need to transfer smolt intoseawater during the smolt window.

[0093] Standard smolts that are newly placed in ocean netpens are notable to grow optimally during their first 40-60 day interval in seawaterbecause of the presence of osmotic stress that delays their feeding.This interval of osmotic adaptation prevents the smolts from takingadvantage of the large number of degree days present immediately aftereither spring or fall placement. The combination of the presence of thesmolt window together with delays in achieving optimal smolt growthprolong the growout interval to obtain market size fish. This isparticularly problematic for S0's since the timing of their harvest iscomplicated by the occurrence of grilsing in maturing fish that areexposed to reductions in ambient photoperiod.

[0094] Methods

[0095] The following examples refer to APS Process I and APS Process IIthroughout. APS stands for “AquaBio Products Sciences®), L.L.C.” APSProcess I is also referred to herein as “SUPERSMOLT™ I Process” or“Process I.” An “APS Process I” fish or smolt refers to a fish or smoltthat has undergone the steps of APS Process I. An APS Process I smolt isalso referred to as a “SUPERSMOLT™ I” or a “Process I” smolt. Likewise,APS Process II is also referred to herein as “SUPERSMOLT™ II Process” or“Process II.” An “APS Process II” fish or smolt refers to a fish orsmolt that has undergone the steps of APS Process II. An APS Process IIsmolt is also referred to as a “SUPERSMOLT™ II” or a “Process II” smolt.

[0096] APS Process I: Pre-adult anadromous fish (this includes bothcommercially produced S0, S1 or S2 smolts as well as smaller parr/smoltfish) are exposed to or maintained freshwater containing either 2.0-10.0mM Calcium and 0.5-10.0 mM Magnesium ions. This water is prepared byaddition of calcium carbonate and/or chloride and magnesium chloride tothe freshwater. Fish are fed with feed pellets containing 7%(weight/weight) NaCl. See Example 8 for further details regarding thefeed. Fish are exposed to or maintained in this regimen of water mixtureand feed for a total of 30-45 days, using standard hatchery caretechniques. Water temperatures vary between 10-16° C. Fish are exposedto a constant photoperiod for the duration of APS Process I. Afluorescent light is used for the photoperiod.

[0097] APS Process II: Pre-adult anadromous fish (this includes bothcommercially produced S0, S1 or S2 smolts as well as smaller parr/smoltfish) are exposed to or maintained in freshwater containing 2.0-10.0 mMCalcium and 0.5-10.0 mM Magnesium ions. This water is prepared byaddition of calcium carbonate and/or chloride and magnesium chloride tothe freshwater. Fish are fed with feed pellets containing 7%(weight/weight) NaCl and either 2 gm or 4 gm of L-Tryptophan per kg offeed. See Example 8 for further details regarding the feed. Fish areexposed to or maintained in this regimen of water mixture and feed for atotal of 30-45 days using standard hatchery care techniques. Watertemperatures vary between 10-16° C. Fish are exposed to a constantphotoperiod for the duration of APS Process II. A fluorescent light isused for the photoperiod.

[0098] Results and Discussion:

[0099] Section I: Demonstration of the Benefits of the APS Process I forAtlantic Salmon, Trout and Arctic Char.

[0100] Demonstration of the Benefits of the APS Process I for AtlanticSalmon:

[0101] APS Process I increases the survival of small Atlantic Salmon S2smolt after their transfer to seawater when compared to matchedfreshwater controls. Optimal survival is achieved by using the completeprocess consisting of both the magnesium and calcium water mixture aswell as NaCl diet. In contrast, administration of calcium and magnesiumeither via the food only or without NaCl dietary supplementation doesnot produce results equivalent to APS Process I.

[0102] Table 1 shows data obtained from Atlantic salmon S2 smolts lessthan 1 year old weighing approximately 25 gm. This single group of fishwas apportioned into 4 specific groups as indicated below and each weremaintained under identical laboratory conditions except for thevariables tested. All fish were maintained at a water temperature of9-13° C. and a continuous photoperiod for the duration of theexperiment.

[0103] The control freshwater group that remained in freshwater for theinitial 45 day interval experienced a 33% mortality rate under theseconditions such that only 67% were able to be transferred to seawater.After transfer to seawater, this group also experienced high mortalitywhere only one half of these smolts survived. Inclusion of calcium (10mM) and magnesium (5 mM) within the feed offered to smolt(Ca2+/Mg2+diet) reduced survival as compared to controls both infreshwater (51% vs 67%) as well after seawater transfer (1% vs 50%). Incontrast, inclusion of 10 mM Ca2+ and 5 mM Mg2+ in the freshwater (APSProcess I Water Only) improved smolt survival in APS Process I water aswell as after transfer of smolt to seawater. However, optimal resultswere obtained (99% survival in both the APS Process I water mixture aswell as after seawater transfer) when smolt were maintained in APSProcess I water mixture and fed a diet supplemented with 7% sodiumchloride. TABLE 1 Comparison of the Survival of Atlantic Salmon S2Smolts After Various Treatments Parameter Control Ca2+/Mg2+ APS WaterAPS Water + Sampled Freshwater Diet Only NaCl Diet Starting # of 66 7074 130 fish # of fish 44 36 67 129 % of fish  67%  51%  91%  99%surviving after 45 days in freshwater or APS mixture # of fish 22  2 60128 % of fish  50%  1%  90%  99% surviving 5 days after transfer toseawater

[0104] Application of the APS Process I to the Placement of 70-100 gmsmolts in seawater.

[0105] These data show that use of the APS Process I eliminates the“smolt window” and provides for immediate smolt feeding and significantimprovement in smolt growth rates.

[0106] Experimental Protocol:

[0107] Smolts derived from the St. John strain of Atlantic salmonproduced by the Connors Brothers Deblois Hatchery located inCherryfield, Me., USA were utilized for this large scale test. Smoltswere produced using standard practices at this hatchery and were derivedfrom a January 1999 egg hatching. All smolts were transferred withstandard commercially available smolt trucks and transfer personnel. S1smolt were purchased during Maine's year 2000 smolt window and smoltdeliveries were taken between the dates of Apr. 29, 2000-May 15, 2000.Smolts were either transferred directly to Polar Circle netpens (24 mdiameter) located in Blue Hill Bay Me. (Controls) or delivered to thetreatment facility where they were treated with APS Process I for atotal of 45 days. After receiving the APS Process I treatment, the smoltwere then transported to the identical Blue Hill Bay netpen site andplaced in an adjacent rectangular steel cage (15 m×15 m×5 m) forgrowout. Both groups of fish received an identical mixture of moist (38%moisture) and dry (10% moisture) salmonid feed (Connors Bros). Each ofthe netpens were fed by hand or feed blower to satiation twice per dayusing camera visualization of feeding. Mort dives were performed on aregular basis and each netpen received identical standard care practicesestablished on this salmon farm. Sampling of fish for growth analyseswas performed at either 42 days (APS Process I) or 120 days or greater(Control) fish. In both cases, fish were removed from the netpens andmultiple analyses performed as described below.

[0108] All calculations to obtain feed conversion ratio (FCR) orspecific growth rate (SGR) and growth factor (GF3) were performed usingstandard accepted formulae (Willoughby, S. Manual of Salmonid FarmingBlackwell Scientific, Oxford UK 1999) and established measurements ofdegree days for the Blue Hill Bay site as provided in Table 2 below. Adegree day is a measure of the number of days that a month in which asalmon can grow. It is calculated by multiplying the number of days in amonth by the amount of degrees in Celsius. TABLE 2 Degree days for BlueHill Bay Salmon Aquaculture Site Month Degree Days Jan 60 Feb 30 Mar 15April 120 May 210 June 300 July 390 Aug 450 Sept 420 Oct 360 Nov 240 Dec180

[0109] Table 3 displays data obtained after seawater transfer of ControlS1 smolt. Smolt ranging from 75-125 gm were placed into 3 independentnetpens and subjected to normal farm practices demonstratedcharacteristics typical for present day salmon aquaculture in Maine.Significant mortalities (average 3.3%) were experienced after transferinto cool (10° C.) seawater and full feeding was achieved only after asignificant interval (˜56 days) in ocean netpens. As a result, theaverage SGR and GF3 values for these 3 netpens were 1.09 and 1.76respectively for the 105-121 day interval measured.

[0110] In contrast to the immediate transfer of Control S1 smolt asdescribed above to ocean netpens (Table III), a total of 10,600 S1 smoltpossessing an average size of 63.6 grams were transported on May 11,2000 from the Deblois freshwater hatchery to the research facility.While being maintained in standard circular tanks, these fish were heldfor a total of 45 days at an average water temperature of 11° C. andwere subjected to APS Process I. During this interval, smolt mortalitywas only 64 fish (0.6%). As a matched control for the APS Process Ifish, a smaller group of control fish (n=220) were held under identicalconditions but did not receive the APS Process I treatment. Themortalities of these control fish were minimized by the holdingtemperature of 10° C. and were equivalent to treated smolts prior totransfer to seawater. TABLE 3 Characteristics of St. John S1 smoltsubjected to immediate placement in ocean netpens after transport formthe freshwater hatchery without APS technology (the Control fish) NetpenNumber #17 #18 #10 Total Fish 51,363 43,644 55,570 Mean Date of 5/1/005/5/00 5/14/00 Seawater Transfer Average Size at (117.6) 75-100 75-100Transfer (grams) 100-125 Mortalities after 30 1,785; 3.5% 728; 1.7%2503; 4.5% days (# and % total) Time to achieve full 68 days 48 days 50days feeding after transfer Interval between 121 120 105 netpenplacement and analysis Average size at Analysis Weight (gram) 376.8 ± 74305.80 ± 64 298.90 ± 37.40 Length (cm) 33.4 ± 1.9 28.30 ± 9.0 30.40 ±1.17 Condition Factor (k) 1.02 1.34 1.06 SGR 0.96 1.10 1.17

[0111] During the 45 day interval when S1 smolts were receiving APSProcess I, fish grew an average of 10 grams and thus possessed anaverage weight of 76.6 gm when transferred to an ocean netpen. Theactual smolt transfer to seawater occurring on Jun. 26, 2000 was notablefor the unusual vigor of the smolt that would have normally beenproblematic since this time is well past the normal window for oceanplacement of smolt. The ocean temperature at the time of APS Process Ismolt netpen placement was 15.1° C. In contrast to the counterpart S1smolts subjected to standard industry practices described above, APSProcess I smolts fed vigorously within 48 hours of ocean placement andcontinued to increase their consumption of food during the immediatepost-transfer period. The mortality of APS Process I smolts was low(6.1%) during initial 50 days after ocean netpen placement and twothirds of those mortalities were directly attributable to scale loss andother physical damage incurred during the transfer process itself.

[0112] In contrast, corresponding control fish (held under identicalconditions without APS Process I treatment) did not fare well duringtransfer to the netpen (17% transfer mortality) and did not feedvigorously at any time during the first 20 days after ocean netpenplacement. This smaller number of control fish (176) were held in asmaller (1.5 m×1.5 m×1.5 m) netpen floating within the larger netpencontaining APS Process I smolts. Their mortality post-ocean netpenplacement was very high at 63% within the 51 day interval.

[0113] Both APS Process I and control smolts were fed on a daily basisin a manner identical to that experienced by the Industry Standard Fishshown on Table 3. APS Process I fish were sampled 51 days after theirseawater placement and compared to the Industry Standard smolts shown onTable 2. As shown in Table 4, comparison of their characteristicsreveals dramatic differences between Industry Standard smolts vs APSProcess I. TABLE 4 Comparison of the characteristics of St. John S1 APSProcess I Smolts subjected to APS treatment and then placed in oceannetpens vs corresponding industry standard smolts. Averaged Industry APSProcess I Standard Data from Table Smolts 3 in this Example Total Fish10,600 150,577 Mean Date of Seawater 6/26/00 5/7/00 Transfer AverageSize at Transfer 76.6 95.8 (grams) Mortalities after 30 days 648; 6.1%21,618; 14.3% (# and %) Time to achieve full 48 hrs 56 days Feedingafter transfer Interval between netpen 51 115 placement and analysisAverage size at Analysis 175.48 + 50 327.2 Weight (gram) 262.22 + 3230.7 Length (cm) Condition Factor (k) 0.95 + 0.9 1.14 SGR 1.80 1.09

[0114] In summary, notable differences between APS Process I, Controlsmolt and Industry Standard smolt include:

[0115] 1. The mortalities observed after ocean netpen placement were lowin APS Process I (6.1%) vs Control (63%) despite the that fact thesefish were transferred to seawater 1.5 months after the smolt window andinto a very high (15.1° C.) ocean water temperature. The mortality ofAPS Process I was actually lower than that of Industry Standard smolt(14.3%) transferred to cooler (10° C.) seawater during the smolt window.This characteristic of APS Process I provides for a greater flexibilityin freshwater hatchery operations since placement of APS Process Ismolts are not rigidly confined the conventional “smolt window”currently used in industry practice.

[0116] 2. The APS Process I fish were in peak condition during andimmediately after seawater transfer. Unlike industry standard smolt thatrequired 56 days to reach full feeding, the APS Process I smolts fedvigorously within 48 hours. Moreover, the growth rates (SGR 1.8)demonstrated by APS Process I smolts are significantly greater than bothpublished data for standard smolt during their initial 50 days afterseawater placement (published values (Stradmeyer, L. Is feedingnonstarters a waste of time. Fish Farmer 3:12-13, 1991; Usher, M L, CTalbot and F B Eddy. Effects of transfer to seawater on growth andfeeding in Atlantic'salmon smolts (Salmo salar L.) Aquaculture94:309-326, 1991) for SGR's range between 0.2-0.8). In fact, the growthrates of APS Process I smolts are significantly larger than Industrystandard smolts placed on the same site despite the fact that industrystandard smolt were both larger at the time of seawater placement aswell as the fact that their growth was measured 120 days after seawaterplacement. These data provide evidence that the APS Process I smoltswere not subjected to significant osmoregulatory stress which wouldprevent them from feeding immediately.

[0117] 3. The rapid growth of APS Process I smolts immediately uponocean netpen placement provides for compounding increases in the size ofsalmon as seawater growout proceeds. Thus, it is anticipated that ifIndustry Standard Smolts weighing 112.5 gram were subjected to APSProcess I treatment, placed in ocean netpens and examined at 120 daysafter ocean netpen placement their size would be average 782 graminstead of 377 gram as observed. This provides for more than a doublingin size of fish in the early stages of growout. Such fish would reachmarket size more rapidly as compared to industry standard fish.

[0118]FIG. 6 provides data on the characteristics of APS Process Ismolts after seawater transfer.

[0119] Application of the APS Process I to Atlantic Salmon Pre-adultFish that are Smaller than the Industry Standard “Critical Size” Smolt.

[0120] A total of 1,400 Landcatch/St John strain fingerlings possessingan average weight of 20.5 gram were purchased from Atlantic Salmon ofMaine Inc., Quossic Hatchery, Quossic, Me., USA on Aug. 1, 2000. Thesefingerlings were derived from a egg hatching in January 2000 andconsidered rapidly growing fish. They were transported to the treatmentfacility using standard conventional truck transport. After theirarrival, these fingerlings were first placed in typical freshwatergrowout conditions for 14 days. These fingerlings were then subjected toAPS Process I for a total of 29 days while being exposed to a continuousphotoperiod. The APS Process I were then vaccinated with the LipogenForte product (Aquahealth LTD.) and transported to ocean netpens byconventional truck transport and placed into seawater (15.6° C.) ineither a research ocean netpen possessing both a predator net as well asnet openings small enough (0.25 inch) to prevent loss of these smallerAPS Process I smolts. Alternatively, APS Process I smolts were placed incircular tanks within the laboratory. Forty eight hours after sea watertransfer, APS Process I smolts were begun on standard moist (38%moisture) smolt feed (Connors Bros.) that had been re-pelletized due tothe necessity to provide for smaller size feed for smaller APS Process Ismolts, as compared to normal industry salmon. In a manner identical tothat described for 70 gram smolts above, the mortality, feedconsumption, growth and overall health of these 30 gram APS Process Ismolts were monitored closely.

[0121]FIG. 7 displays the characteristics of a representative sample ofa larger group of 1,209 APS Process I smolts immediately prior to theirtransfer to seawater. These parameters included an average weight of26.6+8.6 gram, length of 13.1+1.54 cm and condition factor of 1.12+0.06.After seawater transfer, APS Process I smolts exhibited a low initialmortality despite the fact that their average body weight is 26-38% ofindustry standard 70-100 gram S0-S1 smolts. As shown in Table 5, APSProcess I smolts mortality within the initial 72 hr after seawaterplacement was 1/140 or 0.07% for the laboratory tank. Ocean netpenmortalities after placement of APS Process I smolts were 143/1069 or13.4%. FIG. 7 shows representative Landcatch/St John strain APS ProcessI smolts possessing a range of body sizes that were transferred toseawater either in ocean netpens or corresponding laboratory seawatertanks. APS Process I smolts possess a wide range of sizes (46.8-5.6grams body weight) with an average body weight of 26.6 gram. TABLE 5Characteristics and survival of APS Landcatch/St. John Supersmolts Iafter their placement into seawater in either an APS laboratory tank orocean netpen. Laboratory Tank Ocean Netpen Total Fish 140 1,069 Date ofSeawater Transfer 9/5/00 (40); 9/12/00 (100) 9/12/00 Average Size atTransfer 26.6 26.6 (gram) Total mortalities after 4 1; 0.7% 143; 13.4%days (# and % total) % mortality of fish 0; 0.0% 4; 0.4% weighing 25 gmand above Time to achieve feeding 48 hrs 72 hrs

[0122]FIG. 8 shows a comparison of the distributions of bodycharacteristics for total group of Landcatch/St John APS Process Ismolts vs. mortalities 72 hr after seawater ocean netpen placement.Length and body weight data obtained from the 143 mortalities occurringafter seawater placement of 1,069 APS Process I smolts were plotted ondata obtained from a 100 fish sampling as shown previously in FIG. 7.Note that the mortalities are distributed among the smaller fish withinthe larger APS Process I netpen population.

[0123] Length and weight measurements for all mortalities collected fromthe bottom of the ocean netpen were compared to the distribution of APSProcess I smolt body characteristics obtained from analysis of arepresentative sample prior shown in FIG. 8. The data show that themortalities occurred selectively amongst APS Process I smolts possessingsmall body sizes such that the mean body weight of mortalities was 54%of the mean body weight of the total transfer population (14.7/27 gramor 54%). Thus, the actual mortality rates of APS Process I smoltsweighing 25-30 gram is 0.4% (4/1069) and those weighing 18-30 gram is2.9% (31/1069).

[0124] Application of APS Process I to Trout Pre-adult Fish that areSmaller than the Industry Standard “Critical Size” Smolt.

[0125] Table 6 displays data on the use of the APS Process I on small(3-5 gm) rainbow trout. Juvenile trout are much less tolerant of abrupttransfers from freshwater to seawater as compared to juvenile Atlanticsalmon. As a result, many commercial seawater trout producers transfertheir fish to brackish water sites located in estuaries or fresh waterlenses or construct “drinking water” systems to provide fresh water fortrout instead of the full strength seawater present in standard oceannetpens. After a prolonged interval of osmotic adaptation, trout arethen transferred to more standard ocean netpen sites to complete theirgrowout cycle. In general, trout are transferred to these ocean sitesfor growout at body weights of approximately 70-90 or 90-120 gram. TABLE6 Comparison of the Survival of Rainbow Trout (3-5 gm) in Seawater AfterVarious Treatments. Percent Survival of Fish¹ Constant 23 Constant 14day Hours Post Constant 14 day Photoperiod + Seawater Control dayPhotoperiod APS Transfer Freshwater Photoperiod APS Process Process 0100 100 100 100 24 0 25 80 99 48 0 70 81 72 40 68 96 30 58 120 30 46Number of 10 20 30 80 Fish Per Experiment

[0126] A total of 140 trout from a single pool of fish less than 1 yrold were divided into groups and maintained at a water temperature of9-13° C. and pH 7.8-8.3 for the duration of the experiment describedbelow. When control freshwater rainbow trout are transferred directlyinto seawater, there is 100% mortality within 24 hr (ControlFreshwater). Exposure of the trout to a constant photoperiod for 14 daysresults in a slight improvement in survival after their transfer toseawater. In contrast, exposure of trout to APS Process I for either 14days or 23 days results in significant reductions in mortalties aftertransfer to seawater such that 30% and 46% of the fish respectively havesurvived after a 5 day interval in seawater. These data demonstrate thatapplication of the APS Process I increases in the survival of pre-adulttrout that are less than 7% of the size of standard “critical size”trout produced by present day industry standard techniques.

[0127] Application of the APS Process I to Arctic Char Pre-adult Fishthat are Smaller than the Industry Standard “Critical Size” Smolt.

[0128] Although arctic char are salmonids and anadromous fish, theirtolerance to seawater transfer is far less as compared to either salmonor trout. FIG. 9 shows the results of exposure of smaller char (3-5 gm)to the APS Process I for a total of 14 and 30 days. All fish shown inFIG. 9 were exposed to a continuous photoperiod. Transfer of char toseawater directly from freshwater results in the death of all fishwithin 24 hr. In contrast, treatment of char with the APS Process I for14 and 30 days produces an increase in survival such that 33% (3/9) or73% (22/30) respectively are still alive after a 3 day exposure. Thesedata demonstrate that the enhancement of survival of arctic char thatare less than 10% of the critical size as defined by industry standardmethods after their exposure to the APS Process I followed by transferto seawater.

[0129]FIG. 9 shows a comparison of survival of arctic char after varioustreatments. A single group of arctic char (3-5 gm were obtained fromPierce hatcheries (Buxton, Me.) and either maintained in freshwater ortreated with the APS Process I prior to transfer to seawater.

[0130] Section II: The Use of the APS Process II to Permit SuccessfulTransfer of 10-30 gram Smolt into Seawater Netpens and Tanks.

[0131] The APS Process II protocol is utilized to treat pre-adultanadromous fish for placement into seawater at an average size of 25-30gm or less. This method differs from the APS Process I protocol by theinclusion of L-tryptophan in the diet of pre-adult anadromous fish priorto their transfer to seawater. APS Process II further improves theosmoregulatory capabilities of pre-adult anadromous fish and providesfor still further reductions in the “critical size” for Atlantic salmonsmolt transfers. In summary, APS Process II reduces the “critical size”for successful seawater transfer to less than one fifth the size of thepresent day industry standard SO smolt.

[0132] Application of APS Process II to Atlantic Salmon Fingerlings:

[0133] St John/St John strain pre-adult fingerlings derived from aJanuary 2000 egg hatching and possessing an average weight of 0.8 gramswere purchased from Atlantic Salmon of Maine Inc. Kennebec Hatchery,Kennebec Me. on Apr. 27, 2000. These fish were transported to thetreatment facility using standard conventional truck transport. Aftertheir arrival, these parr were first grown in conventional flow throughfreshwater growout conditions that included a water temperature of 9.6°C. and a standard freshwater parr diet (Moore-Clark Feeds). On Jul. 17,2000, fingerlings were begun on APS Process II for a total of 49 dayswhile being exposed to a continuous photoperiod. APS Process II smoltswere then vaccinated with the Lipogen Forte product (Aquahealth LTD.) onDay #28 (Aug. 14, 2000) of APS Process II treatment. APS Process IIsmolts were size graded prior to initiating APS Process II as well asimmediately prior to transfer to seawater. St John/St John APS ProcessII smolts were transported to ocean netpens by conventional trucktransport and placed into seawater (15.2° C.) in either a single oceannetpen identical to that described for placement of APS Process I smoltsor into laboratory tanks (15.6° C.) within the research facility.

[0134]FIG. 10 shows representative St. John/St John strain APS ProcessII smolts possessing a range of body sizes were transferred to seawatereither in ocean netpens or corresponding laboratory seawater tanks. Notethat these APS Process II smolts possess a wide range of body weights(3.95-28 gram) that comprised an average body weight of 11.5 gram. FIG.10 shows the characteristics of St. John/St John APS Process II smolts.The average measurements of these St. John/St. John APS Process IIsmolts included a body weight of 11.50+5.6 gram, length of 9.6+1.5 cmand condition factor of 1.19+0.09. The data displayed in Table 7 showsthe outcomes for two groups of APS Process II smolts derived from asingle production pool of fish after their seawater transfer into eitherlaboratory tanks or ocean netpens. Although important variables such asthe water temperatures and transportation of fish to the site ofseawater transfer were identical, these 2 groups of APS Process IIsmolts experienced differential post seawater transfer mortalities after5 days into laboratory tanks (10% mortality) and ocean netpens (37.7%mortality). TABLE 7 Characteristics and survival of APS St. John/St.John SuperSmolts II after their placement into seawater in either alaboratory tank or ocean netpen. Laboratory Tank Ocean Netpen Total Fish100 1,316 Seawater Transfer Date 8/31/00 9/5/00 Water Temperature (° C.)15.6 15.6 Size at Transfer (gram) 11.5 11.5 Total Mortalities after 510; 10% 496; 37.7% days (# and % total) % mortalities weighing 13 0; 0%1; 0.08% grams or greater Time to achieve feeding 48 hrs 48 hrs aftertransfer

[0135] No apparent problems were observed with the smaller (10-30 gram)APS Process II smolts negotiating the conditions that exist within theconfines of their ocean netpen. This included the lack of apparentproblems including the ability to school freely as well as the abilityto swim normally against the significant ocean currents that arecontinuously present in the commercial Blue Hill Bay salmon aquaculturesite. While these observations are still ongoing, these data do notsuggest that the placement and subsequent growth of APS Process IIsmolts in ocean netpens will be comprised because of lack of ability ofthese pre-adult anadromous fish to swim against existing ocean currentsand therefore be unable to feed or develop properly.

[0136]FIG. 11 compares characteristics of survivors and mortalities ofAPS Process II smolts after seawater transfer to either laboratory tanks(FIG. 11A) or ocean netpens (FIG. 11B). FIG. 11A data are derived fromanalyses of 100 APS Process II smolts transferred to seawater tank whereall fish were killed and analyzed on Day 5. In contrast, FIG. 11Bdisplays only mortality data from ocean netpen. In both cases, onlysmaller APS Process II smolts experienced mortality. Note differences inY axis scales of FIGS. 11A-B.

[0137] Comparison of the average body size of those APS Process IIsmolts that survived seawater transfer vs. those APS Process II smoltsthat died shows that unsuccessful APS Process II smolts possessedsignificantly smaller body weights as compared to average body size ofwhole APS Process II smolt transfer group. Thus, the average weight ofmortalities in laboratory tank (5.10+2.2 gram) and ocean netpen(6.46+1.5 gram) are 44% and 56% respectively the value of the averagebody weight possessed by the entire transfer cohort (11.5 gram). Incontrast, the mortalities of APS Process II smolts with body weightsgreater than 13 gram is 0/100 in the laboratory tank and 1/1316 or0.076% for ocean netpens. Together, these data demonstrate that APSProcess II is able to redefine the “critical size” of Atlantic salmonsmolts from 70-100 gram to approximately 13 gram.

[0138] Application of the APS Process II to Rainbow Trout

[0139] Expansion of trout farming has been hampered by several factors.These include the fact that juvenile trout are much less tolerant ofabrupt transfers from freshwater to seawater as compared to juvenileAtlantic salmon. As a result, many commercial seawater trout producerstransfer their fish to brackish water sites located in estuaries orfresh water lenses or construct “drinking water” systems to providefresh water for trout instead of the full strength seawater present instandard ocean netpens. After a prolonged interval of osmoticadaptation, trout are then transferred to more standard ocean netpensites to complete their growout cycle. In general, trout are transferredto these ocean sites for growout at body weights of approximately 70-90or 90-120 gram.

[0140] A total of 2,000 Donaldson strain trout with an average weight of18 gram were obtained from a local commercial hatchery source (Pine TreeTrout Farm, Sanford, Me., USA). They were derived from a December 1999egg hatching and were transferred from freshwater to the APS Process IIat 11-12° C. while being exposed to a continuous photoperiod. The totalduration of APS Process II treatment was 35 days (Jun. 21-Jul. 26,2000). After being vaccinated using Lipogen Forte (Aquahealth LTD),trout were transferred directly to a research netpen containing fullstrength seawater at 15.6° C. using standard transfer procedures asdescribed for Atlantic salmon above. The average weight for the totalgroup of Trout APS Process II was 22.7 gram as shown on Table 8.

[0141] Mortality counts performed identically to those described forAtlantic salmon transfers revealed a total of 513/1190 or 43.1% duringthe initial 5-day interval. The average body weight of these mortalitieswas 15.5+1.5 gram as shown on FIG. 12. In a manner similar to thatdisplayed by Atlantic salmon APS Process I and II smolts, mortalitiesoccurred amongst the smaller trout APS Process II smolts while thelarger fish exhibited little or no deaths. Thus, the average body weightfor the mortality population was 15.5 gram or 68.3% of the value fortotal population of trout transferred to seawater. Feeding of trout wasobserved upon offering moist diet feed at 48 hours after placement infull strength seawater. TABLE 8 Characteristics and Survival ofDonaldson Rainbow Trout SuperSmolts II After Their Direct Placement intoFull Strength Seawater in APS Ocean Netpen. Trout APS SuperSmolts IITotal Fish 1,190 Date of Seawater Transfer 7/25/00 Average Size atTransfer (grams) 22.7 grams Mortalities after 5 days (# and % totla)513; 43.1% Average Size of Morts (grams) 15.5 ± 1.52 Average Size ofSurvivors (grams) 29.35 + 8.3 Time to achieve feeding after transfer 48hours

[0142]FIG. 12 shows a distribution of body weights and lengths amongstmortalities of trout APS Process II smolts during the initial 5 daysafter transfer to ocean netpens. Note that the average weight of these515 mortalities is 15.5+1.5 gram.

[0143] In summary, these data demonstrate that the benefits of thepresent invention are not confined to Atlantic salmon but also occurusing rainbow trout. Application of the APS Process II smolts processhas significantly reduced the “critical size” of rainbow trout fordirect seawater transfer to approximately 30 gram. Moreover, it haseliminated the necessity for the transfer of rainbow trout into brackishwater. Thus, application of the APS Process II promises to greatlyexpand the possible number of sites that can be utilized for fullstrength seawater transfer of rainbow trout.

[0144] Quantitation of Feeding and Growth of APS Process I and II smoltsafter Seawater Transfer:

[0145] Landcatch/St John APS Process I smolts were offered foodbeginning 48 hr after their seawater transfer to either laboratory tanksor ocean netpens. While these APS Process I smolts that were transferredto laboratory tanks began to feed after 48 hr, those fish transferred toocean netpens were not observed to feed substantially until 7 days. Tovalidate these observations, the inventors performed direct visualinspection of the gut contents from a representative sample of 49 APSProcess I smolts 4 days after their seawater transfer to laboratorytanks. A total of 21/49 or 42.9% possessed food within their gutcontents at that time.

[0146] The St John/St John APS Process II smolts fed vigorously whenfirst offered food 48 hrs after their seawater transfer regardless ofwhether they were housed in laboratory tanks or ocean netpens. Anidentical direct analysis of APS Process II smolts gut contentsperformed as described above revealed that 61/83 or 73.5% of fish werefeeding 4 days after transfer to seawater. The vigorous feeding activityof APS Process II smolts in an ocean netpen as well as laboratory tanksoccurred. Taken together, these data suggest that APS Process I and IIsmolts do not suffer from a prolonged (20-40 day) interval of poorfeeding after seawater transfer as is notable for the much largerindustry standard Atlantic salmon smolts not treated with the process.

[0147] Due to the fact that these groups of pre-adult Atlantic salmonsubjected to either APS Process I or APS Process II have beentransferred very recently to seawater, it is not possible to report ontheir ocean netpen growth rates, as shown for larger S1 smolts (Table4). However, APS has quantified the growth rates of identical fishtreated with either APS Process I or II within laboratory seawatertanks. As shown in Table 9, both Atlantic salmon treated with APSProcess I or II grow rapidly during the initial interval after transferto seawater. In contrast to industry standard smolt weighing 70-100grams that eat poorly and thus have little or no growth during theirfirst 20-30 days after transfer to seawater, pre-adult Atlantic salmonreceiving APS Process I or II both exhibited substantial weight gainsand growth despite the fact that they are only 27-38% (APS Process I)and 12-16% (APS Process II) for the critical size of industry standardsmolts. TABLE 9 Comparison of Growth Rates of Pre-adult Atlantic SalmonExposed to either APS Process I or APS Process II and Placed inLaboratory Tanks During Initial Interval After Seawater Transfer APSProcess I APS Process II Number of Fish 140 437 Weight at Placement into26.6 11.50 Seawater Days in Seawater 22 21 Placement Weight 26.6* 13.15*Corrected for Mortalities Weight after Interval in 30.3 15.2 SeawaterWeight Gained in 3.75 2.05 Seawater SGR (% body weight/day) 0.60 0.68FCR 1.27 2.04

Example 3 Exposure of Salmon Smolts to Ca2+ and Mg2+ IncreasesExpression of PVCR.

[0148] In smolts that were exposed to 10 mM Ca²⁺ and 5.2 mM Mg²⁺, theexpression of PVCR was found to increase in a manner similar to that insmolts that are untreated, but are transferred directly to seawater.

[0149] Tissues were taken from either Atlantic salmon or rainbow trout,after anesthesitizing the animal with MS-222. Samples of tissues werethen obtained by dissection, fixed by immersion in 3% paraformaldehyde,washing in Ringers then frozen in an embedding compound, e.g., O.C.T.™(Miles, Inc., Elkahart, Ind., USA) using methylbutane cooled on dry ice.After cutting 8 micron thick tissue sections with a cryostat, individualsections were subjected to various staining protocols. Briefly, sectionsmounted on glass slides were: 1) blocked with goat serum or obtainedfrom the same species of fish, 2) incubated with rabbit anti-CaRantiserum, and 3) washed and incubated with peroxidase-conjugatedaffinity-purified goat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum were visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections were mounted, viewed and photographed by standardlight microscopy techniques. The methods used to produce anti-PVCRantiserum are described below.

[0150] The results are shown in FIGS. 13A-13G, which are a set of sevenphotomicrographs showing immunocytochemistry of epithelia of theproximal intestine of Atlantic salmon smolts using anti-PVCR antiserum,and in FIG. 14, which is a Western blot of intestine of a salmon smoltexposed to Ca2+- and Mg2+-treated freshwater, then transferred toseawater. The antiserum was prepared by immunization of rabbits with a16-mer peptide containing the protein sequence encoded by the carboxylterminal domain of the dogfish shark PVCR (“SKCaR”) (Nearing, J. et al.,1997, J. Am. Soc. Nephrol. 8:40A). Specific binding of the anti-PVCRantibody is indicated by aminoethylcarbazole (AEC) reaction product.

[0151]FIGS. 13A and 13B show stained intestinal epithelia from smoltsthat were maintained in freshwater then transferred to seawater and heldfor an interval of 3 days. Abundant PVCR immunostaining is apparent incells that line the luminal surface of the intestine. The highermagnification (1440×) shown in FIG. 13B displays PVCR protein localizedto the apical (luminal-facing) membrane of intestinal epithelial cells.The pattern of PVCR staining is localized to the apical membrane ofepithelial cells (small arrowheads) as well as membranes in globularround cells (arrows). FIG. 13C shows stained intestinal epithelia from arepresentative smolt that was exposed APS Process I and maintained infreshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days. Note thatthe pattern of PVCR staining resembles the pattern exhibited byepithelial cells displayed in FIGS. 13A and 13B including apicalmembrane staining (small arrowheads) as well as larger globular roundcells (arrows). FIG. 13D shows a 1900× magnification of PVCR-stainedintestinal epithelia from another representative fish that was exposedto the APS Process I and maintained in freshwater containing 10 mM Ca2+and 5.2 mM Mg2+ for 50 days and fed 1% NaCl in the diet. Again, smallarrowhead and arrows denote PVCR staining of the apical membrane andglobular cells respectively. In contrast to the prominent PVCR stainingshown in FIGS. 13A-D, FIGS. 13E (1440×) and 6F (1900×) show staining ofintestinal epithelia from two representative smolt that were maintainedin freshwater alone without supplementation of Ca2+ and Mg2+ or dietaryNaCl. Both 13E and 13F display a marked lack of significant PVCRstaining. FIG. 13G (1440×) shows the lack of any apparent PVCR stainingupon the substitution of preimmune anti-PVCR antiserum on a sectioncorresponding to that shown in FIG. 13A where anti-PVCR antiserumidentified the PVCR protein. The lack of any PVCR staining is a controlto demonstrate the specificity of the anti-PVCR antiserum under theseimmunocytochemistry conditions.

[0152] The relative amount of PVCR protein present in intestinalepithelial cells of freshwater smolts (FIGS. 13E and 13F) was negligibleas shown by the faint staining of selected intestinal epithelial cells.In contrast, the PVCR protein content of the corresponding intestinalepithelial cells was significantly increased upon the transfer of thesesmolts to seawater (FIGS. 13A and 13B). Importantly, the PVCR proteincontent was also significantly increased in the intestinal epithelialcells of smolts maintained in freshwater supplemented with Ca2+ and Mg2+(FIGS. 13C and 13D). The AEC staining was specific for the presence ofthe anti-PVCR antiserum, since substitution of the immune antiserum bythe preimmune eliminated all reaction product from intestinal epithelialcell sections (FIG. 13G).

[0153] To further demonstrate the specificity of the anti-CaR antiserumto recognize salmon smolt PVCRs, FIG. 14 shows a Western blot ofintestinal protein from salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+and fed 1% NaCl in the diet. Portions of the proximal and distalintestine were homogenized and dissolved in SDS-containing buffer,subjected to SDS-PAGE using standard techniques, transferred tonitrocellulose, and equal amounts of homogenate proteins as determinedby both protein assay (Piece Chem. Co, Rocford, Ill.) as well asCoomassie Blue staining were probed for presence of PVCR using standardwestern blotting techniques. The results are shown in the left lane,labeled “CaR”, and shows a broad band of about 140-160 kDa and severalhigher molecular weight complexes. The pattern of PVCR bands is similarto that previously reported for shark kidney (Nearing, J. et al., 1997,J. Am. Soc. Nephrol. 8:40A) and rat kidney inner medullary collectingduct (Sands, J. M. et al., 1997, J. Clin. Invest. 99:1399-1405). Thelane on the right was treated with the preimmune anti-PVCR antiserumused in FIG. 13G, and shows a complete lack of bands. Taken togetherwith immunocytochemistry data shown in FIG. 13, this immunoblotdemonstrates that the antiserum used is specific for detecting the PVCRprotein in salmon.

Example 4 Exposure of Trout Fingerlings to Ca2+ and Mg2+ IncreasesExpression of PVCRs.

[0154] Development of specific ion transport capabilities in epithelialcells of gill, kidney and intestinal tissues are important to survivalof pre-adult anadromous fish if they are to survive transfer toseawater. To determine if alterations in the PVCRs accompanied theincrease in trout fingerling survival in seawater, immunoblotting andimmunocytochemistry was performed on samples from the fingerlings as wasdone for the salmon smolt tissues. The results are shown in FIGS. 15, 16and 19.

[0155]FIG. 15 is an immunoblot of intestinal tissue from troutfingerlings. Anti-CaR antiserum identifies multiple bands that arespecific for PVCR staining as determined by comparison of immune (lanemarked CaR) vs. preimmune (lane marked pre-immune). Prominent amongthese bands includes a broad band of 120-160 kDa, together with largermolecular weight complexes present above these bands from both intestineand gill tissue.

[0156]FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G and 16H are a set of eightphotomicrographs showing immunocytochemistry of epithelia of theproximal intestine of rainbow trout using anti-PVCR antiserum. FIGS.16A, 16C and 16E show samples from trout maintained in freshwater alone,while FIGS. 16B, 16D, 16F, 16G and 16H show samples from troutmaintained in freshwater supplemented with 10 mM Ca2+ and 5.2 mM Mg2+and fed a 1% NaCl diet. Proximal intestinal segments are shown in FIGS.16A-16D, and 16G-16H, while distal intestinal segments are shown inFIGS. 16E-16F. FIGS. 16A-16F were treated with immune rabbit anti-CaRantiserum, washed, and developed with horseradish peroxidase-conjugatedgoat anti-rabbit antiserum using an aminoethylcarbazole (AEC) reaction.While FIGS. 16A, 16C and 16E display little or no PVCR staining, FIGS.16B, 16D and 16F show significant PVCR staining that is present on theapical membrane of cells lining the intestinal lumen (small arrowheads)as well as larger globular round cells (arrows). In contrast to sectionsexposed to immune anti-PVCR antiserum, FIG. 16H was treated withpre-immune rabbit anti-CaR antiserum and thus do not contain the coloredAEC reaction product. These data indicate this method specificallydetects PVCR protein bound to the anti-PVCR antiserum. FIG. 16G wasstained directly with Alcian blue (Sheehan, D. C. et al., 1980, Theoryand Practice of Histochemistry, Battelle Press, Columbus, Ohio, USA) tolocalize mucin-producing epithelial cells that are present in intestine.Note the appearance of cells staining for PVCR protein in FIG. 13D(denoted by small arrows) display a similar morphological appearance tothose stained with Alcian blue in FIG. 13G. These data suggest that PVCRare expressed by mucin producing cells in the intestine where PVCRsignaling actions modulate mucin production in the intestine.

[0157] Immunocytochemistry of intestinal tissue shows that the contentof PVCR protein is different in trout maintained in freshwater alone(FIGS. 16A, 16C and 16E) vs. freshwater supplemented with Ca2+ and Mg2+and trout fed a NaCl supplemented diet (FIGS. 16B, 16D, 16F). Normallyin freshwater, CaR expression is low in either proximal (FIGS. 15A and16C) or distal (FIG. 16E) sections of intestine. However, PVCRexpression is significantly increased in both proximal (FIGS. 16B and15D) and distal segments (FIG. 15F) after exposure to freshwatersupplemented with 10 mM Ca2+ and 5.2 mM Mg2+ and feeding of NaClsupplemented diet.

[0158] While PVCR protein is localized to several regions of multiplecells, the presence of intense staining on the apical membranes ofintestinal epithelial cells (small arrowheads) as well as occasionalrounded cells (large arrowheads) are identical to data localizing PVCRprotein in both the dogfish shark (Nearing, J. et al., 1997, J. Am. Soc.Nephrol. 8:40A), as well as rat kidney inner medullary collecting duct(IMCD) (Sands, J. M. et al., 1997, J. Clin. Invest. 99:1399-1405). Asdescribed above for Atlantic salmon smolts, the apical PVCR in troutintestine is induced by increases in luminal Ca2+ and Mg2+concentrations, and thereby regulates the NaCl-mediated recovery ofwater from intestinal contents. This recovery is important to thesurvival of marine fish (Evans, D. H., 1993, “Osmotic and IonicRegulation,” in: The Physiology of Fishes, ed. D. H. Evans, CRC Press,Boca Raton, Fla., USA, Chapter 11, pp. 315-341), as it replaces osmoticwater losses that occur via the skin and gill.

[0159] The anti-PVCR staining of rounded cells, which are interspersedthroughout the larger intestinal epithelial cells (FIG. 16D) is alsoconsistent with these cells corresponding to mucin-producing cells whichare known to stain intensely with Alcian Blue (Sheehan, D. C. et al.,1980, Theory and Practice of Histochemistry, Battelle Press, Columbus,Ohio, USA) (FIG. 16G).

[0160]FIG. 17 shows a representative immunoblot that compares theoverall levels of PVCR content of protein homogenates prepared from gilltissue of trout using the same anti-PVCR antiserum as described in FIGS.13-15. Prior to dissection and homogenation of gill tissue, trout wereexposed to 1 of 3 different treatments including either freshwater,freshwater with 10 mM calcium and 5.2 mM magnesium with dietary NaClsupplementation or freshwater with dietary NaCl supplementation only. Inthe gill, the anti-PVCR antiserum also identifies a broad 120-140 kDaand a band of large molecular mass (greater than 200 kDa) that aresimilar to those shown in FIGS. 14 and 15. These data are consistentwith molecular masses of CaRs of known structure and similar to thoseobserved in immunoblotting analyses of multiple organisms, including rat(Sands, J. M. et al., 1997, J. Clin. Invest. 99:1399-1405), flounder,and shark (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A). Amoderate level of PVCR expression in gill as defined by PVCR reactivebands occurs when trout are maintained in freshwater (freshwater). Theabundance of PVCR protein is increased when trout are exposed to the APSProcess I (Ca, Mg+NaCl suppl. Feed) as shown in the middle lane (Ca,Mg). In contrast, when trout are maintained in freshwater and fed a NaCldiet without exposure to calcium and magnesium in the freshwater, thereis no change in the overall PVCR staining intensity but rather a shiftof PVCR reactivity from the 120 kDa to the larger 200 kDa highermolecular weight band (lane marked salt). These data demonstrate thatexposure of trout to the APS Process I (freshwater containing 10 mMcalcium, 5 mM magnesium and dietary NaCl supplementation) increases PVCRexpression in gill tissue as compared to freshwater alone. Feeding ofNaCl supplement diet while the trout are maintained in freshwater doesnot produce similar increased expression of the PVCR protein.

Example 5 Immunolocalization of Polyvalent Cation Receptor (PVCR) inMucous Cells of Epidermis and in the Brain of Salmon

[0161] The skin surface of salmonids is extremely important as a barrierto prevent water gain or loss depending whether the fish is located infresh or seawater. Thus, the presence of PVCR proteins in selected cellsof the fish's epidermal layer would be able to “sense” the salinity ofthe surrounding water as it flowed past and provide for the opportunityfor continuous remodeling of the salmonid's skin based on thecomposition of the water where it is located.

[0162] Methods: Samples of the skin from juvenile Atlantic Salmonresident in seawater for over 12 days were fixed in 3% paraformaldehydedissolved in buffer (0.1M NaP04, 0.15M NaCl, 0.3M sucrose pH 7.4),manually descaled, rinsed in buffer and frozen at −80° C. forcryosectioning. Ten micron sections were either utilized forimmunolocalization of PVCR using anti-shark PVCR antiserum or staineddirectly with 1% Alcian Blue dye to localize cells containing acidicglycoprotein components of mucous.

[0163] Results and Discussion: FIG. 18A shows that salmon epidermiscontains multiple Alcian Blue staining cells present in the various skinlayers. Note that only a portion of some larger cells (that containingacidic mucins) stains with Alcian Blue (denoted by the open arrowheads).For purposes of orientation, note that scales have been removed soasterisks denote surface that was previously bathed in seawater. FIG.18B shows immunolocalization of salmon skin PVCR protein that islocalized to multiple cells (indicated by arrowheads) within theepidermal layers of the skin. Note that anti-PVCR staining shows thewhole cell body, which is larger than its corresponding apical portionthat stains with Alcian Blue as shown in FIG. 18A. The presence of boundanti-CaR antibody is indicated by the rose color reaction product.Although formal quantitation has not yet been performed on thesesections, it appears that the number of PVCR cells is less than thetotal number of Alcian Blue positive cells. These data indicate thatonly a subset of Alcian Blue positive cells contain abundant PVCRprotein. FIG. 18C of FIG. 18 shows the Control Preimmune section wherethe primary anti-CaR antiserum was omitted from the staining reaction.Note the absence of rose colored reaction product in the absence ofprimary antibody.

[0164] These data demonstrate the presence of PVCR protein in discreteepithelial cells (probably mucocytes) localized in the epidermis ofjuvenile Atlantic salmon. From this location, the PVCR protein could“sense” the salinity of the surrounding water and modulate mucousproduction via changes in the secretion of mucous or proliferation ofmucous cells within the skin itself. The PVCR agonists (Ca2+, Mg2+)present in the surrounding water activate these epidermal PVCR proteinsduring the interval when smolts are being exposed to the process of thepresent invention. This “preconditioning” of Atlantic salmon smolts bythe process of the present invention is important to increased survivalof smolts after their transfer to seawater.

[0165] Localization of PVCR Protein in Brain of Atlantic Salmon:

[0166] The PVCR protein can been specifically localized to the brainstem area of Atlantic salmon using immunocytochemistry and antibodyraised against a peptide sequence found in the carboxyl terminal of theshark PVCR. These data are consistent with a role for a PVCR in themodulation of endocrine function as well as appetite control in Atlanticsalmon.

[0167] Localization of the expression of calcium receptors to specificregions of the mammalian brain has been determined. While the exactfunctions of mammalian CaRs in many regions of the mammalian brain arestill unknown, several lines of evidence indicate that CaRs canintegrate alterations in systemic calcium, sodium and water metabolismwith modulations in brain function that include differences in thesecretion of hormones such as adrenocorticotrophin (ACTH) from thehypothalamus as well as behavioral changes such as regulation of thirstor eating. Of importance to disclosure findings detailed below, PVCRs(CaRs) roles in alteration of endocrine function, drinking and appetitein anadromous fish undergoing transfer from freshwater to seawater areimportant.

[0168] In mammalian brain, there is prominent CaR expression in thesubfornical organ or SFO. The SFO is a key hypothalamic thirst centerand is believed to play a role in modulation of drinking activity tointegrate body calcium and water homeostasis. Stimulation of drinkingbehavior by systemic hypercalcemia via stimulation of CaRs located inthe SFO is thought to minimize the dehydration produced by alterationsin kidney function that blunt the tubular reabsorption of filtered waterby the kidney. The equivalent SFO area of the fish brain is notpresently been identified.

[0169] In the mammalian brain, there is CaR expression in the pons areaof the brainstem particularly around the area postrema near the thirdventricle. The area postrema is known to be collection of neuronsbelieved to mediate appetite and has been termed the “nausea center”.From this portion of the mammalian brain, neuronal pathways provide forintegration of sensory input from vestibular function (sensing ofbalance) as well as visual input via pathways from optic nerves andtheir respective nuclei in the brain. This region of the brain isbelieved to be intimately involved in the nausea produced byhypercalcemia as well as the administration of opiates to humans. Theequivalent area postrema of the fish brain is not presently identified.

[0170] A combination of physiological and anatomic data provide evidencefor the role of CaRs to integrate a variety of endocrine functions withchanges in the serum calcium levels in humans. Intravenous infusion ofcalcium sufficient to raise serum calcium concentrations causesselective increases in gonadotropic releasing hormones and thyroidreleasing hormone (TRH) as well ACTH that are produced by the anteriorpituitary. The anterior pituitary gland is known to be intimatelyconnected with specific areas of the hypothalamus that express CaRs.

[0171] Increases in the serum calcium concentrations of humans causemultiple alterations in both behavior as well as endocrine function.Thus, hypercalcemia causes increased drinking, decreased foodconsumption and alterations in the circulating levels of specifichypothalamic hormones. As mentioned below, analogous changes in behaviorand circulating hormone levels occur in preadult anadromous fish duringsmoltfication and transfer from freshwater to seawater.

[0172] Methods:

[0173] Whole brains obtained from preadult Atlantic salmon (St. John/StJohn APS Process II smolts) that were subjected to the APS Process IIand transferred to seawater were dissected free of their surroundingsand fixed in 3% paraformaldehyde (PFA) in buffer [identical to otherimmunocytochemistry descriptions]. Eight micron sections were cut,attached to glass slides and processed for immunocytochemistry usingeither nonimmune control antiserum or anti-PVCR of dogfish shark.Specific antibody binding was detected by the rose-colored reactionproduct formed from the action of horseradish peroxidase conjugated goatanti-rabbit secondary antiserum and amino ethylcarbazole. Sections wereviewed and photographed using standard light microscopy techniques.

[0174] Results and Discussion:

[0175] After examining serial sections from multiple preadult Atlanticsalmon (average wt. 10.3 gm), there is consistent localization of PVCRprotein in cells localized in 3 distinct regions of the salmon brain.The first region of PVCR localization is distinct staining of neurons inthe vagal lobe region. The second region of PVCR staining is withinneurons in the commissural nucleus of Cajal. Both of these regions ofsalmon brain are known to represent important nuclei in the gustatory(sensing food and eating) as well as general visceral activitiesincluding esophageal and intestinal motility (processing of food andintestinal contents for nutrient and water reabsorption). Expression ofPVCR protein links alterations in both serum and CNS calciumconcentrations to changes in eating and processing of intestinalcontents important for anadromous fish adaptation to seawater.

[0176] A third site of PVCR localization in salmon brain is the saccusvasculosus where PVCR protein is distributed throughout multiple celltypes. The saccus vasculosus is ovid and localized on the ventralsurface of the brain between the inferior lobes. This structure ishighly vascularized and contains connections between the cerebral spinalfluid and the vascular space. Moreover, neurons present in the saccusvasculosus possess massive nerve projections that tract to thesubependymal region of the thalamus. The saccus vasculosus systemmodulates the function of centers of the posterior tubercle andperiventricular thalamus. These areas of the brain are immediatelyadjacent to the pituitary gland.

[0177] The localization of PVCR protein(s) in the brain of preadultAtlantic salmon provides evidence that PVCR can be involved in a varietyof functions in the central nervous system of anadromous fish in amanner similar to that described above for the mammalian brain. Inparticular, localization of PVCR to nuclei that are part of thegustatory system in Atlantic salmon indicates that PVCR protein isexpressed in neurons that modulate appetite similar to that describedfor the area postrema in mammals. Stimulation of PVCR or alterations inits expression via changes in the serum calcium, magnesium or sodiumconcentrations as demonstrated for Atlantic salmon in this applicationwould then be able to modulate appetite and food consumption.Alternatively, alterations in the cerebral spinal fluid concentration ofthese ions via exchange between the CSF and the vascular system can alsobe involved. Since Atlantic salmon smolt produced by present dayindustry standard methods experience an interval of profound anorexiaafter their transfer to seawater, this well known suppression ofappetite can be mediated through PVCR signaling mechanisms.

[0178] In a similar manner, PVCR signaling pathways can also modulateboth drinking behavior and pituitary hormone secretion. PVCR proteinexpressed in the saccus vasculosus can provide for both the initiationof the drinking of seawater by Atlantic salmon and can be directlyanalogous to increased drinking in mammals caused by hypercalcemia.Increases in serum calcium, magnesium and sodium concentrations producedby transfer of preadult anadromous fish from freshwater to seawater canalso be the stimulus for increased secretion of hypothalamic hormonessuch as ACTH. ACTH stimulates the secretion of cortisol by the adrenalgland in fish. Cortisol is one hormone that has been shown to be amodulator of ion transport activity and involved in modulation of theparr-smolt transformation in anadromous fish. Modulation of pituitaryactivity via connections between the saccus vasculosus, hypothalamus andthe pituitary can modulate these endocrine changes.

Example 7 Serum Level in Fish Exposed to APS Process I or APS Process II

[0179] The data described herein demonstrates that Alterations in theConcentrations of Calcium, Magnesium and NaCl in the Body Fluids ofAnadromous Fish Occur After Seawater Transfer and Excessively HighConcentrations cause or contribute to Post Seawater Transfer Deaths inAnadromous Fish. APS Process II Mimics Seawater Transfer WithoutSubjecting Small Preadult Anadromous Fish to Osmotic Stress. This“Preconditions” Fish thus Allowing Them to be Transferred to Seawater atSignificantly Smaller Sizes and Under Conditions That are Nonpermissiveusing Industry Standard Practices.

[0180] PVCRs are present in multiple tissue locations where PVCRs areexposed to surrounding seawater (gills, skin), luminal contents oftubules (kidney, intestine) as well as internal body fluids (brain,endocrine tissue, muscle). When anadromous fish are transferred fromfresh to seawater there is an abrupt rise in the external waterconcentrations of calcium, magnesium and NaCl. If the fish absorbsincreased amounts of calcium, magnesium and NaCl via drinking or osmosisthen PVCRs located on the apical surfaces of intestinal and kidneyepithelial cells will be exposed to increased amounts of these divalentand monovalent ions. These increases in divalent cation concentrationsoccur since the kidney is the primary excretory organ for divalentcations and the intestine is the major water recovery organ foranadromous fish via the processing of ingested seawater. Important forthis data disclosure is the fact that if the concentrations of calcium,magnesium and NaCl increase in the blood and extracellular fluid offish, then the PVCRs that are bathed in these body fluids will becomestimulated. Alterations in serum calcium and magnesium constitute anactual signaling pathway. In this regard, it is also noteworthy thatthere are a wide range of “normal” values for serum concentrations ofcalcium, sodium, magnesium and chloride in anadromous fish. While it hasbeen recognized that steady state serum concentrations of these ionschange with differing salinities, there has been no recognition thatthese might represent fish with differing PVCR “set points” as describedherein.

[0181] Current production methods for salmonids depend on the attainmentof a “critical size” for preadult fish called smolt to enable them tosurvive the transfer from freshwater to seawater.

[0182] The production of salmonids for aquaculture is dependent on theability for preadult fish to survive direct transfer from freshwater toseawater. For this process to occur, present day industry methods haveidentified a “critical size” for each species of salmonid. Below thiscritical size, many fish are not able to survive the dramaticalterations in water osmolality and ionic composition. Factors thatcontribute to the ability of “critical size” smolt include specificsurface area to volume ratios as well as the maturity of ionic transportand hormonal mechanisms to cope with the new seawater ionic environment.These mechanisms involve coordinated responses from several organsincluding the gill, gastrointestinal tract, kidney, and skin as well asspecific behavioral changes such as the initiation of drinking behaviorafter seawater exposure. The transfer of a fish from a freshwater toseawater environment constitutes a major challenge to theseosmoregulatory systems that are rapidly remodeled to permit itssurvival. The basic osmoregulatory mechanisms and responses are outlinedbriefly on FIG. 19.

[0183] When a fish resides in freshwater, it is surrounded by an aqueousenvironment that possesses a significantly lower ionic and osmoticcontent (Table 11). Due to the osmotic gradient that exists between thebody fluid of the fish and the surrounding environment, the fish isconstantly gaining water that continuously threatens to dilute the moreconcentrated ionic content of the fish's body fluids. As a result, thefreshwater fish do not drink and excrete a copious dilute urine. Toprevent the loss of important body salts into the environment, thegills, gastrointestinal tract as well as kidney tubules engage in activeuptake of ions from either their luminal contents or the surroundingfreshwater. TABLE 11 Comparison of the Ionic¹ and Osmotic² Compositionof Seawater and Freshwater vs Serum (Blood) of Atlantic Salmon³ SeawaterFreshwater Atlantic Salmon Sodium 450  0.3-5 135-185 Calcium 10 0.07-22.5-3.9 Magnesium 50 0.04-3 1.0-2.8 Chloride 513  0.23-10 120-138Sulfate 26 0.05 <0.02 Osmolality 1050    1-20 330-390

[0184] In contrast, when a fish resides in seawater the surroundingaqueous environment possess a significantly larger ionic and osmoticcontent as compared to the fish's own body composition (Table 11). Asshown in FIG. 19, marine salmonids are constantly losing body watercontent to the surrounding seawater. In this regard, both the integrityand permeability of the fish's skin layer are important in reducingthese cutaneous losses to as low as possible. To replace these ongoingwater losses, the fish drinks seawater and processes it in such a way toretain water and only a portion of its constituent ions. Ingestedseawater is processed by epithelial cells lining the gastrointestinaltract. In this process, the intestinal uptake of water and some NaCl bythe fish is permitted while Ca2+ and Mg2+ are either not absorbed orexcreted by kidney tubules. Absorbed NaCl is pumped from the fish's bodyvia gill epithelial cells.

[0185]FIG. 19 compares adaptive changes present in fish in freshwater vsseawater. Specific physiological adaptations present in freshwater fishare shown schematically on the left panel. In contrast, alterations inthese same physiological responses when fish are in seawater are shownon the right.

[0186] It is important for the pre-adult anadromous fish to accomplishall of these adaptative changes rapidly after transfer from freshwaterto seawater. Deployment and maturation of these mechanisms requires thesynthesis of new proteins and remodeling of epithelial cells involved intransepithelial transport. These changes occur in a time scale that willpermit the smolt to survive in its new seawater environment. The smallerthe fish, the larger its surface area/volume ratio. Thus, smaller fishlose their body water more rapidly and have less body water stores tobuffer changes in body ionic composition. As a result, small fishrapidly lose water and they cannot replace this water via drinkingseawater since their ionic removal mechanisms are not mature. As aresult, smaller or nonmature smolts rapidly die of electrolyte and waterimbalances produced by their inability to adapt to the new osmotic andionic environment of seawater. In contrast, larger smolts that arelarger than the “critical size” possess a lower surface area to volumeratio, lose water less rapidly and have more body water to buffer ionicchanges. This larger body size provides them the interval of timenecessary to deploy their more mature ionic transport mechanismsenabling them to survive.

[0187] In smolts that are either less than the critical size or possessimmature physiological ion transport mechanisms, the combination of theosmotic removal of water from their bodies coupled with ingestion of ionrich seawater produces specific alterations in body fluid andelectrolyte composition. These changes include: a decrease in total bodywater content, increases in the concentrations of calcium, magnesium andsodium chloride. Abnormally high concentrations of these monovalent anddivalent cations causes a wide range of specific changes in organ andcellular functions including alterations in cellular metabolism andnerve conduction, depression of normal nervous system and muscleactivity as well as cessation of normal ingestion of food and itsdigestion. The abnormal behavior and appearance of highly stresspre-moribund fish after seawater transfer are actually attributable tothe physiological effects of elevated ions including calcium andmagnesium within the body fluids of the fish.

[0188] As described herein, measurements of serum calcium, magnesium andsodium confirm these data as well as demonstrate that the presentinvention causes a preconditioning of physiological and ionic transportmechanisms permitting the successful seawater transfer of preadultanadromous fish that are significantly smaller than the critical size asdefined by present day industry standard methods.

[0189] Methods:

[0190] Blood was obtained from fish (salmon and trout) via venipunctureinto the caudal sinus and prevented from coagulation by the addition oflithium heparin. The blood was centrifuged at 4,000 rpm for 10 minutesand the resulting serum collected and stored until assay. Calcium andmagnesium concentrations of 2 microliter aliquots of serum werequantified using calcium and magnesium assay kits (Kit #595, #587 SigmaAldrich, St Louis, Mo.) and Na was determined by commercial testing(NorDx Laboratories, Scarborough, Me.) using a Hitachi 747 analyzer.

[0191] Results and Discussion:

[0192] The APS Process II Mimics Exposure to Seawater Without thePresence of a Large Osmotic Gradient Between the Fish's Body Fluids andSurrounding Hypertonic Seawater.

[0193]FIG. 20 shows the changes in serum calcium concentrations injuvenile trout (average body weight approximately 30 gm) subjected toseawater transfer either directly from treshwater or after exposure tovarious components of APS Process II. The average steady state serumcalcium concentration in these trout maintained in freshwater is2.72+0.16 mM. In contrast, transfer of trout to seawater results in asignificant rise in serum calcium to approximately 3.80 mM within theinitial 24 hr after seawater transfer. This increase in serum calcium issustained for an interval of approximately 108 hr (4.5 days) but thendeclines to a slightly lower average concentration of 3.20+0.42 mM by126 hr. Thus, internal PVCRs are exposed to a rise in serum calcium upontransfer of freshwater trout to seawater. The aquatic PVCRs wouldactually sense and respond to alterations in calcium this concentrationrange. Thus, the increase in serum calcium (a PVCR agonist) likelyconstitutes a signal for the initiation of multiple PVCR-activatedprocesses in various organs to permitting the survival of juvenile troutin seawater.

[0194] Placement of trout in the water mixture of the present inventionwhich contains 3 mM calcium and 1 mM magnesium, and feeding the trout astandard freshwater diet (Moore Clarke Feeds) results in no significantincreases in serum calcium as compared to serum calcium values for troutmaintained in freshwater despite the presence of a net inward gradientof calcium from external water mixture (3 mM) to internal body fluids(2.72 mM). Moreover, serum calcium concentrations of trout maintained inthe water mixture are not changed by alterations of the ambientphotoperiod from a normal (10 hr daylight; 14 hr darkness) to continuousdaylight exposure.

[0195]FIG. 21 shows increases in serum calcium concentrations induced byfeeding trout maintained in water mixture (3 mM calcium, 1 mM magnesium)a standard freshwater pelleted diet containing additional 1% sodiumchloride (w/w). Feeding of NaCl supplemented diet began immediatelyafter determination of baseline serum calcium concentrations at timezero. Note that serum calcium concentrations became elevated after aninterval of 24 hr. Data points shown represent a total of 5 or moreindependent determinations from a single representative experiment.Values at 24 hr and 72 hr are significantly (p<0.05) increased ascompared to the value at zero time.

[0196] In contrast, the feeding of trout maintained in the water mixtureof the present invention with the identical standard feed except withthe addition of 1% NaCl (weight/weight) produces a significant increasein serum calcium concentrations within 24 hr (FIG. 21). This increase inthe serum calcium concentrations of trout mimics the rise produced bytransfer of trout into seawater(compare FIG. 20 vs. 21). This effect ofdietary NaCl to increase serum calcium levels likely occurs because thefish is obligated to excrete this excess NaCl that it has ingested.Ingestion of this excess NaCl activates the fish's drinking behaviorthereby causing it to ingest water mixture containing 3 mM calcium andthereby increases its body fluid calcium content via the intestinalabsorption of calcium. Ingestion of 1% NaCl alone does not alter serumcalcium concentrations. Thus, the serum calcium concentration of troutmaintained in freshwater (2.72+0.43 n=6) was not altered significantlyafter consumption of feed containing 1% NaCl (w/w) for as long as 30days (2.37+0.25 n=5). These data provide a demonstration that thisprotocol is necessary to achieve increase the serum calciumconcentrations in anadromous fish.

[0197] Transfer of Larger Atlantic Salmon Smolts Raised in Freshwaterthat Possess the Industry Standard “Critical Size” to Seawater Raisestheir Serum Calcium and Sodium Concentrations:

[0198] The data displayed in FIG. 20 shows that the mean serum calciumconcentration increases by approximately 40% when trout are transferredfrom freshwater to seawater. The magnitude of this increase isassociated with significant trout mortality (approximately 30-40%) dueto osmoregulatory failure in these fish that are smaller than the“critical size” for trout. In contrast, the magnitude of increases inserum calcium concentrations is smaller (approximately 30% increase)when larger Atlantic salmon smolts that possess the critical size 60-70gms are transferred to seawater (FIGS. 22A-B). During this same intervalafter seawater transfer, serum sodium concentrations in these same fishincrease by approximately 17%. Data derived from both trout (FIG. 21)and salmon (FIGS. 22A-B) were only collected from fish that exhibited novisible signs of stress (i.e. stressed fish exhibit body discoloration,bizarre swimming behavior or markedly decreased activity levels) duringthis experiment.

[0199]FIG. 22 shows alterations in serum calcium (FIG. 22A) and sodium(FIG. 22B) after seawater transfer of S1 Atlantic salmon smolt thatpossess the critical size as defined by standard present day practices.Each data point represents the mean±S.D of 5-10 independentdeterminations

[0200] FIGS. 23A-B show post seawater transfer values for serum calcium,magnesium and sodium obtained from a cohort of 80 S1 Atlantic salmonsmolt identical to that shown in FIG. 22 after a total of 45 days oftreatment with APS Process I (3 mM Ca²⁺, 1 mM Mg²⁺ in water) and 7% NaCldietary supplement in food. Note that the initial serum calcium in fishexposed to APS Process I is slightly larger (2.5 vs 3.0 mM) and changesin serum concentrations of calcium and sodium are similar to thosedisplayed in FIG. 22. Moreover, calcium and magnesium do not undergodramatic increases during the initial 120 hr interval as these fish aretransferred from calcium/magnesium water mixture to seawater. Incontrast, the serum sodium concentration increases approximatley 12%(178.8 mM from 158.0 mM) within the first 24 hr.

[0201] Taken together, these data shown in FIGS. 19-23 demonstrate thatincreases in both the serum calcium and sodium occur after transfer ofpreadult anadromous fish from freshwater to seawater. Moreover, theoverall expression of PVCR protein increases in specific cells involvedin this osmoregulatory response such as intestine. Since PVCRs arecapable of responding to alterations of both calcium and sodium withinthese concentrations ranges, these data indicate that a new “set point”for PVCR activity is established after transfer of fish to seawater.

[0202] The data shown in FIGS. 21-23 demonstrate that treatment ofpreadult anadromous fish with APS Process I causes increases in theserum calcium concentrations of the fish that mimic those produced bytheir transfer from freshwater to seawater. Exposure of the fish to thecombination of calcium and magnesium in the water and NaCl in the feedcauses increased calcium intake that mirrors the drinking of hypertonicseawater without the accompanying osmotic stress. Thus, the PVCRs in theAPS Process I fish have been “preconditioned” by their exposure tocalcium and magnesium and, as a result, the fish is more readily able toadapt to seawater when it is subsequently transferred to it.

[0203] Anadromous Fish Exhibiting Visible Symptoms of Stress AfterTransfer to Seawater Possess Elevated Serum Values of Calcium and/orMagnesium. The Inability of Fish to Excrete These Ions is the MajorCause for Their Death After Seawater Transfer:

[0204] When pre-adult anadromous fish are transferred to seawater eitherdirectly from freshwater or after exposure to the APS Process I, someportion of the total number of fish are often unable to adapt to thedramatic differences in osmolality and ionic composition betweenfreshwater and seawater and die of resulting electrolyte imbalances.Observations that include tracking of fish that will ultimately expirewithin a short time interval (24-120 hr) after seawater transferdemonstrates that they begin to exhibit visible signs of high levels ofstress including alterations in their normal light silver bodycoloration to a darker duskier hue as well as displaying of bizarreswimming behavior or markedly decreased activity levels 24-72 hr beforetheir death.

[0205] Comparison of serum calcium, magnesium and sodium concentrationsfrom control nonstressed fish vs fish exhibiting signs of high levels ofstress show that serum ion concentrations in stressed fish aresignificantly higher as compared to control (Table 12). TABLE 12Comparison of serum concentrations of juvenile Atlantic salmon and troutin seawater judged by visual inspection as either nonstressed orstressed fish. Serum Concentrations in mM Calcium Magnesium IndustryStandard Juvenile Trout Nonstressed Fish 4.03 ± 0.71 (n = 49) Not DoneStressed Fish 4.58 ± 0.78** (n = 63) Not Done APS Treated AtlanticSalmon Nonstressed Fish 3.74 ± 0.52 2.40 ± 0.77 (n = 15) Stressed fish3.97 ± 0.66 4.07 ± 0.60** (n = 16)

[0206] These signs of high stress are directly referable to abnormallyelevated concentrations of calcium, magnesium and sodium ions within thebody fluids of the fish. Thus, preadult anadromous fish that are unableto excrete excess divalent cations as well as process seawater toreplace body water that is lost via osmosis die from the consequences ofelectrolyte imbalances. Anadromous fish below the critical size are notable to rapidly adapt to the new osmotic environment of seawater and dieas a result.

[0207] Exposure of Preadult Atlantic Salmon Fish Below the “CriticalSize” as Defined by Present Day Industry Standard Methods to the APSProcess II Prevents the Lethal Elevations of Serum Calcium, Magnesiumand Sodium and Thus Allows Successful Seawater Transfer of FishPossessing Very Small Body Weights:

[0208] Pre-adult Atlantic salmon of the St John/St John strain werebegun on the APS Process II including a water mixture (3 mM Ca2+ and 1mM Mg2+) as well as feed containing a combination of 7% NaCl and 2 gm/kg(w/w) L-Tryptophan (APS Process II) for a total of 49 days while beingexposed to a continuous photoperiod. These small, but treated pre-adultAtlantic salmon (termed APS Process II smolts) were then placed intoseawater into either a single ocean netpen or into laboratory tanks(15.6° C.) within the research facility. FIG. 24 compares the bodycharacteristics of APS Process II smolts that adapted successfully toseawater vs APS Process II smolts from the same group that were unableto adapt to seawater and died.

[0209] As shown in FIG. 24, only those Atlantic salmon preadult fishtreated with the APS Process II with the smallest body weights(approximately 10%) experienced post seawater mortalities after 5 daysinto laboratory tanks. Comparison of the average body size of 90%surviving APS Process II smolts vs. those 10% APS Process II smolts thatdied shows that unsuccessful APS Process II smolts possessed smallerbody weights (5.10+2.2 gm) as compared to average body size of whole APSProcess II transfer group (11.5+5.65 gm). Thus, the critical size forthese APS Process II smolts is approximately 13 gm. This critical bodysize is only 13-18.6% (13/70-100) that of the critical size definedpreviously by industry standard techniques. Thus, these data show thatthe use of the Process II has reduced the “critical size” of Atlanticsalmon parr/smolt by over 80%.

[0210] Quantitation of serum calcium, magnesium and sodiumconcentrations in APS Process II smolts that have successfully made thetransition from calcium/magnesium water mixture to seawater is shown inFIG. 25. It is noteworthy that serum concentrations of calcium,magnesium or sodium did not change despite the fact that the averagebody size of these APS Process II smolts is less than 20% (11.5/60.5 gm) of the normal “critical size” for Atlantic Salmon smolts producedusing present day industry standard methods. FIG. 25 shows that neitherserum calcium, magnesium or sodium concentrations increase dramaticallyas would be expected from data shown in FIG. 20 and Table 2 as well asdata published previously. Treatment of industry standard Atlanticsalmon smolt/parr with APS Process II results in no dramatic increasesin the concentrations of any ions measured above despite thesignificantly smaller body size of APS Process II smolts, as compared tolarge industry standard smolts. Comparison of data shown in FIGS. 22A-B(Industry standard S1 smolts), FIG. 23 (Industry standard S1 smoltstreated with APS Process I vs FIG. 25 (preadult salmon less than 20% ofthe industry standard critical size) also reveals that these serumconcentrations for the smaller APS Process II smolts are comparable tothose displayed by the larger industry standard S1 smolts. In summary,these data show that preadult Atlantic salmon treated with APS ProcessII do not exhibit dramatic changes in their body composition of calcium,magnesium and sodium despite their significantly smaller size. This lackof alterations in the concentrations of these ions greatly reducesstress in these fish and permits them to adapt to seawater readily.

Example 8 The Feed

[0211] There are two general methods to prepare feed for consumption byfish as part of APS Process I and II. These two processes involve eitherreformulation of feed or addition of a concentration solution forabsorption by the feed followed by a top dressing for palatability. Thisdisclosure describes the methodology to prepare feed using each of these2 methods.

[0212] Methods:

[0213] Feed Manufacture for Salmon Experiments

[0214] To reformulate feed, the ingredients are as follows: Base Dietwas made using the following ingredients and procedure: 30% Squid(liquefied in blender), 70% Corey Aquafeeds flounder diet (powderized inblender). Ingredients were blended into a semi moist “dough” ball. Otheringredients including NaCl or PVCR active compounds were blended intothe base diet by weight according to what the experiment called for.Moore Clark standard freshwater salmonid diet (sizes 1.2, 1.5.2.0, 2.5,and 3.5 mm) can also be used. A top dressing was applied to the pelletssuch that top dressing is composed of 4% of the weight of the Base Diet.Top dressing is composed of 50% krill hydrolysate (Specialty MarineProducts Ltd.) and 50% Menhaden fish oil. The top dressing is added forpalatability and sealing of added ingredients.

[0215] Other ingredients can include NaCl, MgCl2, CaCl2 or L-Tryptophanthat are added by weight to the base diet by weight.

[0216] Preparation of Feed Containing 7% (weight/weight) NaCl:

[0217] For the APS Process I: Solid sodium chloride or NaCl apportionedat a ratio of 7% of the weight of the Moore Clark standard freshwatersalmonid diet weight was added to a volume of tap water approximately3-4 times the weight of NaCl. The mixture was heated to 60-70° C. withmixing via use of a magnetic stirring bar to dissolve salt. The NaClsolution was then poured into a hand held sprayer and applied to theMoore Clark standard freshwater salmonid diet that is tumbling inside ofa 1.5 cubic meter motorized cement mixer. After absorption of the NaClrich solution, the wetted Moore Clark standard freshwater salmonid dietis spread out thinly on window screening and placed in an enclosed racksystem equipped with a fan and 1500 watt heater to expedite dryingprocess. After drying for approximately 6 hr, the dried NaCl-richpellets are returned to the cement mixer and a top dressing is applied.The feed is stored at room temperature until use.

[0218] Preparation of Feed Containing 7% (weight/weight) NaCl+PVCRAgonist (Tryptophan) For the APS Process II: Solid sodium chloride orNaCl apportioned at a ratio of 7% of the weight of the Moore Clarkstandard freshwater salmonid diet weight was added to a volume of tapwater approximately 3-4 times the weight of NaCl. The mixture was heatedto 60-70° C. with mixing via use of a magnetic stirring bar to dissolvesalt. USP Grade L-Tryptophan was added to the water at either 2 grams or4 grams for every kg of Moore Clark standard freshwater salmonid dietdepending on formulation need. Dilute hydrochloric acid was added to thewater with mixing until the tryptophan was dissolved and the pH ofsolution was approximately 4.0. The NaCl+Tryptophan solution was thenpoured into a hand held sprayer and was then applied to the Moore Clarkstandard freshwater salmonid diet tumbling inside a cement mixer. Afterabsorption of the NaCl+Tryptophan solution, the wetted Moore Clarkstandard freshwater salmonid diet is then spread out thinly on windowscreening and placed in an enclosed rack system equipped with a fan and1500-watt heater to expedite drying process. After drying forapproximately 6 hr, the dried NaCl/Tryptophan-rich pellets are thenreturned to the cement mixer and a top dressing is applied. The feed isstored at room temperature until use.

Example 9 DNA and Putative Protein Sequences from Partial Genomic Clonesof Polyvalent Cation Receptor Protein Amplified by PCR from the DNA of 8Species of Anadromous Fish

[0219] These data provide the partial genomic sequences derived from thePVCR gene in 8 species of anadromous fish. Each of these nucleotidesequences is unique and thus could be used as a unique probe to isolatethe full-length cDNA from each species. Moreover, this DNA fragmentcould form the basis for a specific assay kit(s) for detection of PVCRexpression in various tissues of these fish.

[0220] The PVCR has been isolated in several species of salmon, char andtrout. Sequences of mammalian CaRs together with the nucleotide sequenceof SKCaR (FIGS. 28A-B) were used to design degenerate oligonucleotideprimers to highly conserved regions in the extracellular domain ofpolyvalent cation receptor proteins using standard methodologies (See GM Preston, Polymerase chain reaction with degenerate oligonucleotideprimers to clone gene family members, Methods in Mol. Biol. Vol. 58Edited by A. Harwood, Humana Press, pages 303-312, 1993). Using theseprimers, cDNA or genomic DNA from various fish species representingimportant commercial products are amplified using standard PCRmethodology. Amplified bands are then purified by agarose gelelectrophoresis and ligated into appropriate plasmid vector that istransformed into a bacterial strain. After growth in liquid media,vectors and inserts are purified using standard techniques, analyzed byrestriction enzyme analysis and sequenced where appropriate. Using thismethodology, nucleotide sequences were amplified.

[0221] To generate the data displayed in FIGS. 26 and 27, DNA wasisolated from muscle samples of each of the species indicated usingstandard published techniques. DNA was then amplified using polymerasechain reaction (PCR) methodology including 2 degenerate PCR primers(DSK-F3 and DSK-R4; SEQ ID NO:22 and 23). Amplified DNAs were thenpurified by agarose gel electrophoresis, subcloned into plasmid vectors,amplified, purified and sequenced using standard methods.

[0222]FIG. 26 shows an aligned genomic DNA sequences of 594 nucleotidesfor 8 anadromous fish species, each of which codes for an identicalregion of the PVCR protein. Note that each nucleotide sequence derivedfrom each specific species is unique. However, alterations in the DNAsequences of these genes often occur at common specific nucleotideswithin each sequence of 594 nucleotides.

[0223]FIG. 27 shows aligned corresponding predicted protein sequencesderived from genomic nucleotide sequences displayed in FIG. 26. Notethat only 3 alterations in the amino acid sequence of this portion ofthe PVCR occur as a consequence of alterations in the nucleotidesequence as shown in FIG. 26. All of these changes (Ala to Val; Arg toLys; and Cys to Tyr) are known as “conservative” substitutions of aminoacids in that they preserve some combination of the relative size,charge and hydrophobicity of the peptide sequence.

[0224] All cited references, patents, and patent applications areincorporated herein by reference in their entirety. Also, companionpatent application Ser. No. 09/687,373, entitled “Growing Marine Fish inFresh Water,” filed on Oct. 12, 2000; patent application Ser. No.09/687,476, entitled “Methods for Raising Pre-adult Anadromous Fish,”filed on Oct. 12, 2000; patent application Ser. No. 09/687,372, entitled“Methods for Raising Pre-adult Anadromous Fish,” filed on Oct. 12, 2000;Provisional Patent Application No. 60/240,392, entitled “PolyvalentCation Sensing Receptor Proteins in Aquatic Species,” filed on Oct. 12,2000; Provisional Patent Application No. 60/240,003, entitled“Polyvalent Cation Sensing Receptor Proteins in Aquatic Species,” filedon Oct. 12, 2000, are all hereby incorporated by reference in theirentirety. Additionally, application Ser. No. 09/162,021, filed on Sep.28, 1998, International PCT application No. PCT/US97/05031, filed onMar. 27, 1997, and application Ser. No. 08/622,738 filed Mar. 27, 1996,all entitled, “Polycation Sensing Receptor in Aquatic Species andMethods of Use Thereof” are all hereby incorporated by reference intheir entirety.

[0225] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes can be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1 23 1 594 DNA Atlantic Salmon 1 cttggcatta tgctctgtgc tgggggtattcttgacagca ttcgtgatgg gagtgtttat 60 caaatttcgc aacaccccaa ttgttaaggccacaaacaga gagctatcct acctcctcct 120 gttctcactc atctgctgtt tctccagttccctcatcttc attggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgggataagtttt gttctctgca tctcctgcat 240 cctggtaaaa actaaccgag tacttctagtgttcgaagcc aagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagttcctgttagtg ttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaatgctcctccg gcgagctaca ggaaccatga 420 cattgatgag ataattttca ttacatgcaatgagggctct atgatggcgc ttggcttcct 480 aattgggtac acatgcctgc tggcagccatatrcttcttc tttgcattta aatcacgaaa 540 actgccagag aactttactg aggctaagttcatcaccttc agcatgctca tctt 594 2 199 PRT Atlantic Salmon Xaa=any aminoacid 2 Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met 15 10 15 Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn20 25 30 Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser35 40 45 Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu50 55 60 Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile65 70 75 80 Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys IlePro 85 90 95 Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gln Phe LeuLeu 100 105 110 Val Phe Leu Phe Thr Phe Val Gln Val Met Ile Cys Val ValTrp Leu 115 120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp IleXaa Asp Glu 130 135 140 Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met MetAla Leu Gly Phe 145 150 155 160 Leu Ile Gly Tyr Thr Cys Leu Leu Ala AlaIle Xaa Phe Phe Phe Ala 165 170 175 Phe Lys Ser Arg Lys Leu Pro Glu AsnPhe Thr Glu Ala Lys Phe Ile 180 185 190 Thr Phe Ser Met Leu Ile Phe 1953 594 DNA Arctic Char 3 cttggcatta tgctctgtgc tgggggtatt cttgacagcattcgtgatgg gagtgtttat 60 cagatttcgc aacaccccaa ttgttaaggc cacaaacagagagctatcct acctcctcct 120 gttctcactc atctgctgtt tctccagctc cctcatcttcattggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgg gataagttttgttctctgca tctcctgcat 240 cctggtcaaa actaaccgag tacttctagt gttcgaagccaagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagtt cctgttggtgttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaa tgctcctccggcgagctaca ggaaccatga 420 cattgatgag ataattttca ttacatgcaa tgagggctctatgatggcgc tcggcttcct 480 aattgggtac acatgcctgc tggcagccat atgcttcttctttgcattta aatcacgaaa 540 actgccagag aactttaccg aggctaagtt catcaccttcagcatgctca tctt 594 4 199 PRT Arctic Char Xaa = Any amino acid 4 Leu AlaLeu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met 1 5 10 15 GlyVal Phe Ile Arg Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn 20 25 30 ArgGlu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser 35 40 45 SerSer Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu 50 55 60 ArgGln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile 65 70 75 80Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro 85 90 95Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu 100 105110 Val Phe Leu Phe Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu 115120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Xaa Asp Glu130 135 140 Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu GlyPhe 145 150 155 160 Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys PhePhe Phe Ala 165 170 175 Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr GluAla Lys Phe Ile 180 185 190 Thr Phe Ser Met Leu Ile Phe 195 5 593 DNATrout 5 ttggcattat gctctgtgct gggggtattc ttgacagtat tcgtgatgggagtgtttatc 60 agatttcgca acaccccaat tgttaaggcc acaaacagag agctatcctacctcctcctg 120 ttctcactta tctgctgttt ctccagctcc ctcatcttca ttggtgaaccccaggactgg 180 acatgccgtc tacgccagcc tgcattcggg ataagttttg ttctctgcatctcctgcatc 240 ctggtcaaaa ctaaccgagt acttctagtg ttcgaagcaa agatccccaccagtctccat 300 cgtaagtggt gggggctaaa cttgcagttc ctgttggtgt tcctgttcacatttgtgcaa 360 gtgatgatat gtgtggtctg gctttacaat gctcctccgg cgagctacaggaaccatgac 420 attgatgaga tcattttcat tacatgcaat gagggctcta tgatggcgcttggcttccta 480 attgggtaca catgcctgct ggcagccata tgcttcttct ttgcatttaaatcacgaaaa 540 ctgccagaga attttaccga ggctaagttc atcaccttca gcatgctcatctt 593 6 199 PRT Trout Xaa = Any amino acid 6 Leu Ala Leu Cys Ser ValLeu Gly Val Phe Leu Thr Val Phe Val Met 1 5 10 15 Gly Val Phe Ile ArgPhe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn 20 25 30 Arg Glu Leu Ser TyrLeu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser 35 40 45 Ser Ser Leu Ile PheIle Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu 50 55 60 Arg Gln Pro Ala PheGly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile 65 70 75 80 Leu Val Lys ThrAsn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro 85 90 95 Thr Ser Leu HisArg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu 100 105 110 Val Phe LeuPhe Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu 115 120 125 Tyr AsnAla Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Xaa Asp Glu 130 135 140 IleIle Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe 145 150 155160 Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala 165170 175 Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile180 185 190 Thr Phe Ser Met Leu Ile Phe 195 7 594 DNA Chum Salmon 7cttggcatta tgctctgtgc tgggggtatt cttgacagca ttcgtgatgg gagtgtttat 60cagatttcgc aacaccccaa ttgttaaggc cacaaacaga gagctatcct acctcctcct 120gttctcactt atctgctgtt tttccagctc cctcatcttc attggtgaac cccaggactg 180gacatgccgt ctacgccagc ctgcattcgg gataagtttt gttctctgca tctcctgcat 240cctggtcaaa actaaccgag tacttctagt gttcgaagca aagatcccca ccagtctcca 300tcgtaagtgg tgggggctaa acttgcagtt cctgttggtg ttcctgttca catttgtgca 360agtgatgata tgtgtggtct ggctttacaa tgctcctccg gcgagctaca ggaaccatga 420cattgatgag atcattttca ttacatgcaa tgagggctct atgatggcgc ttggcttcct 480aattgggtac acatgcctgc tggcagccat atgcttcttc tttgcattta aatcacgaaa 540actgccagag aattttaccg aggctaagtt catcaccttc agcatgctca tctt 594 8 197PRT Chum Salmon 8 Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr AlaPhe Val Met 1 5 10 15 Gly Val Phe Ile Arg Phe Arg Asn Thr Pro Ile ValLys Ala Thr Asn 20 25 30 Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu IleCys Cys Phe Ser 35 40 45 Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp TrpThr Cys Arg Leu 50 55 60 Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu CysIle Ser Cys Ile 65 70 75 80 Leu Val Lys Thr Asn Arg Val Leu Leu Val PheGlu Ala Lys Ile Pro 85 90 95 Thr Ser Leu His Arg Lys Trp Trp Gly Leu AsnLeu Gln Phe Leu Leu 100 105 110 Val Phe Leu Phe Thr Phe Val Gln Val MetIle Cys Val Val Trp Leu 115 120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr ArgAsn His Asp Ile Asp Glu Ile 130 135 140 Ile Phe Ile Thr Cys Asn Glu GlySer Met Met Ala Leu Gly Phe Leu 145 150 155 160 Ile Gly Tyr Thr Cys LeuLeu Ala Ala Ile Cys Phe Phe Phe Ala Phe 165 170 175 Lys Ser Arg Lys LeuPro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr 180 185 190 Phe Ser Met LeuIle 195 9 594 DNA Coho Salmon 9 cttggcatta tgctctgtgc tgggggtattcttgacagya ttcgtgatgg gagtgtttat 60 cagatttcgc aacaccccaa ttgttaaggccacaaacaga gagctatcct acctcctcct 120 gttctcactt atctgctgtt tctccagctccctcatcttc attggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgggataagtttt gttctctgca tctcctgcat 240 cctggtcaaa actaaccgag tacttctagtgttcgaagca aagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagttcctgttggtg ttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaatgctcctccg gcgagctaca ggaaccatga 420 cattgatgag atcattttca ttacatgcaatgagggctct atgatggcgc ttggcttcct 480 aattgggtac acatgcctgc tggcagccatatgcttcttc tttgcattta aatcacgaaa 540 actgccagag aattttacmg aggctaagttcatcaccttc agcatgctca tctt 594 10 197 PRT Coho Salmon Xaa= Any AminoAcid 10 Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Xaa Phe Val Met1 5 10 15 Gly Val Phe Ile Arg Phe Arg Asn Thr Pro Ile Val Lys Ala ThrAsn 20 25 30 Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys PheSer 35 40 45 Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys ArgLeu 50 55 60 Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser CysIle 65 70 75 80 Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala LysIle Pro 85 90 95 Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gln PheLeu Leu 100 105 110 Val Phe Leu Phe Thr Phe Val Gln Val Met Ile Cys ValVal Trp Leu 115 120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His AspIle Asp Glu Ile 130 135 140 Ile Phe Ile Thr Cys Asn Glu Gly Ser Met MetAla Leu Gly Phe Leu 145 150 155 160 Ile Gly Tyr Thr Cys Leu Leu Ala AlaIle Cys Phe Phe Phe Ala Phe 165 170 175 Lys Ser Arg Lys Leu Pro Glu AsnPhe Thr Glu Ala Lys Phe Ile Thr 180 185 190 Phe Ser Met Leu Ile 195 11594 DNA King Salmon 11 cttggcatta tgctctgtgc tgggggtatt cttgacagcattcgtgatgg gagtgtttat 60 cagatttcgc aacaccccaa ttgttaaggc cacaaacagagagctatcct acctcctcct 120 gttctcactt atctgctgtt tttccagctc cctcatcttcattggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgg gataagttttgttctctgca tctcctgcat 240 cctagtcaaa actaaccgag tacttctagt gttcgaagcaaagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagtt cctgttggtgttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaa tgctcctccagcgagctaca ggaatcatga 420 cattgatgag atcattttca ttacatgcaa tgagggctctatgatggcgc ttggcttcct 480 aattgggtac acgtgcctgc tggcagccat atgcttcttctttgcattta aatcacgaaa 540 actgccagag aattttaccg aggctaagtt cattaccttcagcatgctca tctt 594 12 197 PRT King Salmon 12 Leu Ala Leu Cys Ser ValLeu Gly Val Phe Leu Thr Ala Phe Val Met 1 5 10 15 Gly Val Phe Ile ArgPhe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn 20 25 30 Arg Glu Leu Ser TyrLeu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser 35 40 45 Ser Ser Leu Ile PheIle Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu 50 55 60 Arg Gln Pro Ala PheGly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile 65 70 75 80 Leu Val Lys ThrAsn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro 85 90 95 Thr Ser Leu HisArg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu 100 105 110 Val Phe LeuPhe Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu 115 120 125 Tyr AsnAla Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile 130 135 140 IlePhe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu 145 150 155160 Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe 165170 175 Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr180 185 190 Phe Ser Met Leu Ile 195 13 594 DNA Pink Salmon 13 cttggcattatgctctgtgc tgggggtatt cttgacagct ttcgtgatgg gagtgtttat 60 cagatttcgcaacaccccaa ttgttaaggc cacaaacaga gagctatcct acctcctcct 120 gttctcacttatctgctgtt tttccagctc cctcatcttc attggtgaac cccaggactg 180 gacatgccgtctacgccagc ctgcattcgg gataagtttt gttctctgca tctcctgcat 240 cctggtcaaaactaaccgag tacttctagt gttcgaagca aagatcccca ccagtctcca 300 tcgtaagtggtgggggctaa acttgcagtt cctgttggtg ttcctgttca catttgtgca 360 agtgatgatatgtgtggtct ggctttacaa tgctcctccg gcgagctaca ggaaccatga 420 cattgatgagatcattttca ttacatgcaa tgagggctct atgatggcgc ttggcttcct 480 aattgggtacacatgcctgc tggcagccat atgcttcttc tttgcattta aatcacgaaa 540 actgccagagaattttactg aggctaagtt catcaccttc agcatgctca tctt 594 14 197 PRT PinkSalmon 14 Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe ValMet 1 5 10 15 Gly Val Phe Ile Arg Phe Arg Asn Thr Pro Ile Val Lys AlaThr Asn 20 25 30 Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys CysPhe Ser 35 40 45 Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr CysArg Leu 50 55 60 Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile SerCys Ile 65 70 75 80 Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu AlaLys Ile Pro 85 90 95 Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu GlnPhe Leu Leu 100 105 110 Val Phe Leu Phe Thr Phe Val Gln Val Met Ile CysVal Val Trp Leu 115 120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn HisAsp Ile Asp Glu Ile 130 135 140 Ile Phe Ile Thr Cys Asn Glu Gly Ser MetMet Ala Leu Gly Phe Leu 145 150 155 160 Ile Gly Tyr Thr Cys Leu Leu AlaAla Ile Cys Phe Phe Phe Ala Phe 165 170 175 Lys Ser Arg Lys Leu Pro GluAsn Phe Thr Glu Ala Lys Phe Ile Thr 180 185 190 Phe Ser Met Leu Ile 19515 594 DNA Sockeye Salmon 15 cttggcatta tgctctgtgc tgggggtatt cttgacagcattcgtgatgg gagtgtttat 60 cagatttcgc aacaccccaa ttgttaaggc cacaaacagagaactatcct acctcctcct 120 gttctcactt atctgctgtt tttccagctc cctcatcttcattggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgg gataagttttgttctctgca tctcctgcat 240 cctagtcaaa actaaccgag tacttctagt gttcgaagcaaagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagtt cctgttggtgttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaa tgctcctccagcgagctaca ggaatcatga 420 cattgatgag ataattttca ttacatgcaa tgagggctctatgatggcgy ttggcttcct 480 aattgggtac acgtgcctgc tggcagccat atgcttcttctttgcattta aatcacgaaa 540 actgccagag aattttacag aggctaagtt catcaccttcagcatgctca tctt 594 16 197 PRT Sockeye Salmon Xaa=Any Amino Acid 16 LeuAla Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met 1 5 10 15Gly Val Phe Ile Arg Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn 20 25 30Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser 35 40 45Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu 50 55 60Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile 65 70 7580 Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro 85 9095 Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu 100105 110 Val Phe Leu Phe Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu115 120 125 Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp GluIle 130 135 140 Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Xaa GlyPhe Leu 145 150 155 160 Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys PhePhe Phe Ala Phe 165 170 175 Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr GluAla Lys Phe Ile Thr 180 185 190 Phe Ser Met Leu Ile 195 17 4134 DNADogfish Shark 17 aattccgttg ctgtcggttc agtccaagtc tcctccagtg caaaatgagaaatggtggtc 60 gccattacag gaacatgcac tacatctgtg ttaatgaaat attgtcagttatctgaaggt 120 tattaaaatg tttctgcaag gatggcttca cgagaaatca attctgcacgttttcccatt 180 gtcattgtat gaataactga ccaaagggat gtaacaaaat ggaacaaagctgaggaccac 240 gttcaccctt tcttggagca tacgatcaac cctgaaggag atggaagacttgaggaggaa 300 atggggattg atcttccagg agttctgctg taaagcgatc cctcaccattacaaagataa 360 gcagaaatcc tccaggcatc ctctgtaaac gggctggcgt agtgtggcttggtcaaggaa 420 cagagacagg gctgcacaat ggctcagctt cactgccaac tcttattcttgggatttaca 480 ctcctacagt cgtacaatgt ctcagggtat ggtccaaacc aaagggcccagaagaaagga 540 gacatcatac tgggaggtct cttcccaata cactttggag tagccgccaaggatcaggac 600 ttaaaatcga gaccggaggc gacaaaatgt attcggtaca attttcgaggcttccgatgg 660 ctccaggcga tgatattcgc aattgaagag attaacaaca gtatgactttcctgcccaat 720 atcaccctgg gatatcgcat atttgacacg tgtaacaccg tgtccaaggcgctagaggca 780 acactcagct ttgtggccca gaacaaaatc gactcgctga acttagatgagttctgtaac 840 tgctctgacc atatcccatc cacaatagca gtggtcgggg caaccgggtcaggaatctcc 900 acggctgtgg ccaatctatt gggattattt tacattccac aggtcagctatgcctcctcg 960 agcaggctgc tcagcaacaa gaatgagtac aaggccttcc tgaggaccatccccaatgat 1020 gagcaacagg ccacggccat ggccgagatc atcgagcact tccagtggaactgggtggga 1080 accctggcag ccgacgatga ctatggccgc ccaggcattg acaagttccgggaggaggcc 1140 gttaagaggg acatctgtat tgacttcagt gagatgatct ctcagtactacacccagaag 1200 cagttggagt tcatcgccga cgtcatccag aactcctcgg ccaaggtcatcgtggtcttc 1260 tccaatggcc ccgacctgga gccgctcatc caggagatag ttcggagaaacatcaccgat 1320 cggatctggc tggccagcga ggcttgggcc agctcttcgc tcattgccaagccagagtac 1380 ttccacgtgg tcggcggcac catcggcttc gctctcaggg cggggcgtatcccagggttc 1440 aacaagttcc tgaaggaggt ccaccccagc aggtcctcgg acaatgggtttgtcaaggag 1500 ttctgggagg agaccttcaa ctgctacttc accgagaaga ccctgacgcagctgaagaat 1560 tccaaggtgc cctcgcacgg accggcggct caaggggacg gctccaaggcggggaactcc 1620 agacggacag ccctacgcca cccctgcact ggggaggaga acatcaccagcgtggagacc 1680 ccctacctgg attatacaca cctgaggatc tcctacaatg tatacgtggccgtctactcc 1740 attgctcacg ccctgcaaga catccactct tgcaaacccg gcacgggcatctttgcaaac 1800 ggatcttgtg cagatattaa aaaagttgag gcctggcagg tcctcaaccatctgctgcat 1860 ctgaagttta ccaacagcat gggtgagcag gttgactttg acgatcaaggtgacctcaag 1920 gggaactaca ccattatcaa ctggcagctc tccgcagagg atgaatcggtgttgttccat 1980 gaggtgggca actacaacgc ctacgctaag cccagtgacc gactcaacatcaacgaaaag 2040 aaaatcctct ggagtggctt ctccaaagtg gttcctttct ccaactgcagtcgagactgt 2100 gtgccgggca ccaggaaggg gatcatcgag ggggagccca cctgctgctttgaatgcatg 2160 gcatgtgcag agggagagtt cagtgatgaa aacgatgcaa gtgcgtgtacaaagtgcccg 2220 aatgatttct ggtcgaatga gaaccacacg tcgtgcatcg ccaaggagatcgagtacctg 2280 tcgtggacgg agcccttcgg gatcgctctg accatcttcg ccgtactgggcatcctgatc 2340 acctccttcg tgctgggggt cttcatcaag ttcaggaaca ctcccatcgtgaaggccacc 2400 aaccgggagt tgtcctacct gctgctcttc tccctcatct gctgcttctccagctcgctc 2460 atcttcatcg gcgagcccag ggactggacc tgtcggctcc gccaaccggcctttggcatc 2520 agcttcgtcc tgtgcatctc ctgcatcctg gtgaagacca accgggtgctgctggtcttc 2580 gaggccaaga tccccaccag cctccaccgc aagtgggtgg gcctcaacctgcagttcctc 2640 ctggtcttcc tctgcatcct ggtgcaaatc gtcacctgca tcatctggctctacaccgcg 2700 cctccctcca gctacaggaa ccatgagctg gaggacgagg tcatcttcatcacctgcgac 2760 gagggctcgc tcatggcgct gggcttcctc atcggctaca cctgcctcctcgccgccatc 2820 tgcttcttct tcgccttcaa gtcccgtaag ctgccggaga acttcaacgaggctaagttc 2880 atcaccttca gcatgttgat cttcttcatc gtctggatct ccttcatccccgcctatgtc 2940 agcacctacg gcaagtttgt gtcggccgtg gaggtgattg ccatcctggcctccagcttc 3000 gggctgctgg gctgcattta cttcaacaag tgttacatca tcctgttcaagccgtgccgt 3060 aacaccatcg aggaggtgcg ctgcagcacg gcggcccacg ccttcaaggtggcggcccgg 3120 gccaccctcc ggcgcagcgc cgcgtctcgc aagcgctcca gcagcctgtgcggctccacc 3180 atctcctcgc ccgcctcgtc cacctgcggg ccgggcctca ccatggagatgcagcgctgc 3240 agcacgcaga aggtcagctt cggcagcggc accgtcaccc tgtcgctcagcttcgaggag 3300 acaggccgat acgccaccct cagccgcacg gcccgcagca ggaactcggcggatggccgc 3360 agcggcgacg acctgccatc tagacaccac gaccagggcc cgcctcagaaatgcgagccc 3420 cagcccgcca acgatgcccg atacaaggcg gcgccgacca agggcaccctagagtcgccg 3480 ggcggcagca aggagcgccc cacaactatg gaggaaacct aatccaactcctccatcaac 3540 cccaagaaca tcctccacgg cagcaccgtc gacaactgac atcaactcctaaccggtggc 3600 tgcccaacct ctcccctctc cggcactttg cgttttgctg aagattgcagcatctgcagt 3660 tccttttatc cctgattttc tgacttggat atttactagt gtgcgatggaatatcacaac 3720 ataatgagtt gcacaattag gtgagcagag ttgtgtcaaa gtatctgaactatctgaagt 3780 atctgaacta ctttattctc tcgaattgta ttacaaacat ttgaagtatttttagtgaca 3840 ttatgttcta acattgtcaa gataatttgt tacaacatat aaggtaccacctgaagcagt 3900 gactgagatt gccactgtga tgacagaact gttttataac atttatcattgaaacctgga 3960 ttgcaacagg aatataatga ctgtaacaaa aaaattgttg attatcttaaaaatgcaaat 4020 tgtaatcaga tgtgtaaaat tggtaattac ttctgtacat taaatgcatatttcttgata 4080 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcgg cccgacagcaacgg 4134 18 199 PRT Dogfish Shark 18 Leu Thr Ile Phe Ala Val Leu GlyIle Leu Ile Thr Ser Phe Val Leu 1 5 10 15 Gly Val Phe Ile Lys Phe ArgAsn Thr Pro Ile Val Lys Ala Thr Asn 20 25 30 Arg Glu Leu Ser Tyr Leu LeuLeu Phe Ser Leu Ile Cys Cys Phe Ser 35 40 45 Ser Ser Leu Ile Phe Ile GlyGlu Pro Arg Asp Trp Thr Cys Arg Leu 50 55 60 Arg Gln Pro Ala Phe Gly IleSer Phe Val Leu Cys Ile Ser Cys Ile 65 70 75 80 Leu Val Lys Thr Asn ArgVal Leu Leu Val Phe Glu Ala Lys Ile Pro 85 90 95 Thr Ser Leu His Arg LysTrp Val Gly Leu Asn Leu Gln Phe Leu Leu 100 105 110 Val Phe Leu Cys IleLeu Val Gln Ile Val Thr Cys Ile Ile Trp Leu 115 120 125 Tyr Thr Ala ProPro Ser Ser Tyr Arg Asn His Glu Leu Glu Asp Glu 130 135 140 Val Ile PheIle Thr Cys Asp Glu Gly Ser Leu Met Ala Leu Gly Phe 145 150 155 160 LeuIle Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala 165 170 175Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile 180 185190 Thr Phe Ser Met Leu Ile Phe 195 19 23 PRT Artificial Sequence 23-merpeptide 19 Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg GluGlu 1 5 10 15 Ala Glu Glu Arg Asp Ile Cys 20 20 22 PRT ArtificialSequence peptide 20 Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg GluGlu Ala Glu 1 5 10 15 Glu Arg Asp Ile Cys Ile 20 21 17 PRT ArtificialSequence peptide 21 Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly AspAsp Leu Pro 1 5 10 15 Cys 22 28 DNA Artificial sequence primer 22tgtcktggac ggagccctty ggratcgc 28 23 31 DNA Artificial Sequence primer23 ggckggratg aargakatcc aracratgaa g 31

What is claimed is:
 1. A method of determining whether a pre-adultanadromous fish is ready for transfer to seawater; said methodcomprising assessing the amount of PVCR expression in the pre-adultanadromous fish, wherein an increased level of expression of at leastone PVCR, as compared to a control, indicates that the pre-adultanadromous fish is ready for transfer to seawater.
 2. The method ofclaim 1, further comprising: (a) contacting an anti-PVCR antibody to asample taken from the pre-adult anadromous fish under conditionssufficient for the formation of a complex between the antibody and thePVCR; and (b) detecting the formation of the complex.
 3. The method ofclaim 1, wherein detecting the level of expression of the PVCRcomprises: (a) hybridizing a nucleic acid sequence having a detectablelabel to the nucleic acid sequence of the PVCR of a sample taken fromthe pre-adult anadromous fish, under conditions sufficient for thehybridization thereof; and (b) detecting the hybridization.
 4. A methodof determining whether a pre-adult anadromous fish is ready for transferto seawater; said method comprises: (a) contacting an anti-PVCR antibodyto a sample taken from the pre-adult anadromous fish under conditionssufficient for the formation of a complex between the antibody and thePVCR; and (b) detecting the level of formation of the complex; whereinan increased level of expression of at least one PVCR, as compared to acontrol, indicates that the pre-adult anadromous fish is ready fortransfer to seawater.
 5. A method of determining whether a pre-adultanadromous fish is ready for transfer to seawater; said methodcomprises: (a) hybridizing a nucleic acid sequence having a detectablelabel to the nucleic acid sequence of the PVCR of a sample taken fromthe pre-adult anadromous fish, under conditions sufficient for thehybridization thereof; and (b) detecting the level of hybridization;wherein an increased level of hybridization of at least one PVCR, ascompared to a control, indicates that the pre-adult anadromous fish isready for transfer to seawater.
 6. A method of determining whether apre-adult anadromous fish is ready for transfer to seawater, wherein thepre-adult anadromous fish is subjected to a freshwater environmenthaving at least one PVCR modulator, and feed having at least about 1%NaCl by weight; said method comprising assessing the amount of PVCRexpression in the pre-adult anadromous fish, wherein an increased levelof expression, as compared to a control, indicates that the pre-adultanadromous fish is ready for transfer to seawater.
 7. The method ofclaim 6, further comprising: (a) contacting an anti-PVCR antibody to asample comprising gill, skin, intestine, kidney, brain or muscle; underconditions sufficient for the formation of a complex between theantibody and the PVCR; and (b) detecting the formation of the complex.8. The method of claim 6, wherein detecting the level of expression ofthe PVCR comprises: (a) hybridizing a nucleic acid sequence having adetectable label to the nucleic acid sequence of the PVCR of a sampletaken from the pre-adult anadromous fish, under conditions sufficientfor the hybridization thereof; and (b) detecting the hybridization.
 9. Akit for determining whether a pre-adult anadromous fish is ready fortransfer to seawater; said kit comprises an anti-PVCR antibody.
 10. Akit for determining whether a pre-adult anadromous fish is ready fortransfer to seawater; said kit comprises a nucleic acid sequence havinga detectable label that can hybridize to nucleic acid of an aquaticPVCR.
 11. A method of determining whether a salmon is ready for transferto seawater; said method comprising assessing the amount of PVCRexpression in the salmon, wherein an increased level of expression of atleast one PVCR, as compared to a control, indicates that the salmon isready for transfer to seawater.
 12. The method of claim 11, furthercomprising: (a) contacting an anti-PVCR antibody to a sample taken fromthe salmon under conditions sufficient for the formation of a complexbetween the antibody and the PVCR; and (b) detecting the formation ofthe complex.
 13. The method of claim 11, wherein detecting the level ofexpression of the PVCR comprises: (a) hybridizing a nucleic acidsequence having a detectable label to the nucleic acid sequence of thePVCR of a sample taken from the salmon, under conditions sufficient forthe hybridization thereof; and (b) detecting the hybridization.
 14. Amethod of determining whether a salmon is ready for transfer toseawater, wherein the salmon is subjected to a freshwater environmenthaving at least one PVCR modulator, and feed having at least about 1%NaCl by weight; said method comprising assessing the amount of PVCRexpression in the salmon, wherein an increased level of expression, ascompared to a control, indicates that the salmon is ready for transferto seawater.
 15. The method of claim 14, further comprising: (a)contacting an anti-PVCR antibody to a sample comprising gill, skin,intestine, kidney, brain or muscle; under conditions sufficient for theformation of a complex between the antibody and the PVCR; and (b)detecting the formation of the complex.
 16. The method of claim 14,wherein detecting the level of expression of the PVCR comprises: (a)hybridizing a nucleic acid sequence having a detectable label to thenucleic acid sequence of the PVCR of a sample taken from the salmon,under conditions sufficient for the hybridization thereof; and (b)detecting the hybridization.